Photo-acoustic detection device and method

ABSTRACT

An example system for detecting an analyte in a sample of a bodily fluid comprises a test chamber having at least one sidewall and configured to contain at least a portion of a bodily fluid sample, an excitation electromagnetic energy source configured to direct an energy source into the test chamber through the at least one sidewall and to induce a thermoelastic expansion in the one or more analytes, and a sensor configured to detect said thermoelastic expansion in the bodily fluid sample in the test chamber, the sensor configured to measure changes in optical reflectance that result from the thermoelastic expansion.

REFERENCE TO RELATED APPLICATION AND PRIORITY CLAIM

This application claims priority under 35 U.S.C. §120 and is acontinuation in part of co-pending application Ser. No. 11/827,346,filed on Jul. 11, 2007, which in turn claims priority under 35 U.S.C.§119 from provisional application Ser. No. 60/819,941, which was filedon Jul. 11, 2006.

FIELD OF THE INVENTION

A field of the invention is medical testing. One aspect of the inventionconcerns devices and methods for the detection of analytes in a bodilyfluid sample, with an example being circulating tumor cells in a bloodsample.

BACKGROUND

Detection of analytes in bodily fluid samples is widely used formedicinal and other purposes. Applications include detection ofpathogens, proteins, or other chemical compounds in blood, urine, bile,saliva, or other bodily fluids. Some example applications include drugscreening, detection of disease, detection of a particular protein, andthe like. By way of one particular example, detection of circulatingtumor cells (CTCs) in human blood and lymph systems has the potential toaid clinical decision making in the treatment of cancer. The presence ofCTCs may signify the onset of metastasis, indicate relapse, or may beused to monitor disease progression.

Present techniques and devices for the detection of CTCs have limits.Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is a techniquethat is used in practice. This technique involves the analysis of RNA.The technique takes significant time and involves a number of stepsrequiring the expertise of technicians to conduct assays. With therequirement of such expertise comes the potential for technician error.Freezing, culturing, assaying, etc. in the RT-PCR technique take bothtime and expertise. Results are also not immediately available to atreating physician.

Laser flow cytometry also has the potential to analyze samples for CTCs.However, CTC detection is evolving research area and optimal detectiontechniques are still a work-in-process. The technique, when and if it isperfected, however, remains one that shares some of the drawback toRT-PCR, e.g., skilled technician involvement and delays in obtaining andinterpreting results.

Powerful diagnostic tools permit rapid and accurate evaluation. Criticalto the treatment of cancer is the early stage detection of the onset ofmetastasis or relapse, and the monitoring of disease progression and theresponse of the disease to an ongoing course or treatment. Havingaccurate information about metastasis can provide a treating physicianwith the opportunity to be more effective and address the particularphase of the disease indicated by the metastasis. Accurately and rapidlydetecting the presence of CTCs has the potential to advance the state ofcancer diagnosis and treatment.

SUMMARY

An example method for detecting an analyte in a sample of a bodily fluidcomprises the steps of exposing the bodily fluid sample toelectromagnetic energy to cause a thermoelastic expansion in theanalyte, and detecting a photoacoustic signal in the sample that resultsfrom the thermoelastic expansion.

An example system for detecting one or more analytes in a bodily fluidcomprises a test chamber having at least one sidewall and configured tocontain a bodily fluid test sample, an electromagnetic energy sourceconfigured to direct an energy source into said test chamber throughsaid at least one sidewall and to induce a thermoelectric expansion inthe one or more analytes, and a sensor configured to detect thethermoelastic expansion in the test sample.

An additional example apparatus is a photo-acoustic metastasis detectiondevice comprising a container for containing a fluid sample to be testedfor the presence or absence of circulating tumor cells, a laser tosubject the fluid sample to light that would induce a photo-acousticreaction in circulating tumor cells or a marker attached thereto, and anacoustic sensor to detect a photo-acoustic reaction induced in the fluidsample contained in said container.

An additional example system for detecting an analyte in a sample of abodily fluid comprises a test chamber having at least one sidewall andconfigured to contain at least a portion of a bodily fluid sample, anexcitation electromagnetic energy source configured to direct an energysource into the test chamber through the at least one sidewall and toinduce a thermoelastic expansion in the one or more analytes, and asensor configured to detect said thermoelastic expansion in the bodilyfluid sample in the test chamber, the sensor configured to measurechanges in optical reflectance that result from the thermoelasticexpansion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart illustrating one example method of the invention;

FIG. 2 is a flowchart illustrating a second example method of theinvention;

FIG. 3 is a schematic illustration of an example system of the inventionand is also useful to illustrate an example method of the invention;

FIG. 4 illustrates the test cell of the system of FIG. 3, with FIG. 4(B)illustrating the cell when viewed along the line 4B-4B of FIG. 4(A) inthe direction shown;

FIG. 5 illustrates the test cell of the system of FIG. 3;

FIG. 6 schematically illustrates detection of a thermoelastic eventusing the sensor of the system of FIG. 3;

FIG. 7 illustrates data resultant from practice of an example system andmethod of the invention, with FIG. 7(A) data for a test sample includinglive melanoma and FIG. 7(B) data for a test sample including RPMIculture medium and no melanoma;

FIG. 8 is a schematic illustration of an alternate example flow cell;

FIG. 9 is a schematic illustration of the flow cell of FIG. 8 viewedalong the line 9-9 of FIG. 8 in the direction shown;

FIG. 10 is a schematic illustration of an alternate apparatus of theinvention;

FIG. 11 illustrates a portion of FIG. 10 in greater detail;

FIG. 12 shows data useful to illustrate the embodiment of FIG. 10;

FIG. 13 is example data generated by an example apparatus of theinvention;

FIG. 14 is example data generated by an example apparatus of theinvention;

FIG. 15 is example data generated by an example apparatus of theinvention;

FIG. 16 is example data generated by an example apparatus of theinvention;

FIG. 17 is a schematic of an example invention embodiment, and,

FIG. 18 is a schematic of an additional example invention embodiment.

DETAILED DESCRIPTION

Before example embodiments of the invention are discussed, it will beappreciated that the present invention includes methods as well assystems. For example, a method of the invention may include steps ofusing a system of the invention, and a system of the invention when usedmay be useful to carry out steps of a method of the invention.Accordingly, it will be appreciated that in describing a method of theinvention description of a system may also be provided. Additionally,when describing a system of the invention description of a method of theinvention may be made. For example, it will be appreciated that whendescribing details of a system of the invention description issimultaneously being provided of one or more methods of the invention,and vice versa.

The present invention is directed to methods and systems for detectingan analyte in a bodily fluid sample. As used herein, the term “analyte”is intended to be broadly interpreted as being a chemical, biological orother substance or material of interest. By way of example, an analytemay be a protein, a pathogen such as one or more cancer tumor cells, achemical compound, or the like. Also, as used herein the term “bodilyfluid” is intended to be broadly interpreted as meaning a liquid samplecontaining some fluid that originated in a body. Examples include bodilyfluid samples contain blood, a blood plasma, urine, bile, saliva, semen,sperm, breast milk, cerebrospinal fluid, intracellular fluid, and thelike. Importantly, as used herein the term “bodily fluid sample” is notlimited to the fluid obtained from the body alone, but is also intendedto include samples prepared using the fluid and a diluting fluid orcarrier fluid. By way of example, a sample prepared by suspending abodily fluid such as white blood cells in a saline solution isencompassed by the term “bodily fluid sample.”

In order to best describe various example embodiments of the invention,description of general embodiments (and accordingly a general apparatus)is initially provided. FIG. 1 is a flowchart illustrating one examplemethod of the invention. A bodily fluid sample is exposed to anelectromagnetic energy source to cause a thermoelastic expansion in theanalyte contained in the sample. Block 10. The bodily fluid sample maybe any of the particular samples discussed above, including, forexample, urine, blood or saliva. Also, it may be, for example, a testsample that includes one of these materials diluted, suspended, or otherpresent together with a carrier fluid. A carrier fluid may be a solventor a diluting fluid, with examples including aqueous solutions, salinesolution, and the like.

The electromagnetic energy source may be any suitable energy sourceuseful to cause a thermoelastic expansion. Examples include microwaves,visible and ultraviolet light, and the like. One example energy sourceis laser light. A thermoelastic expansion occurs when a material absorbsenergy, is heated, and expands as a result. A photoacoustic signalresults.

The method of FIG. 1 next includes a step of using an acoustic sensor todetect the acoustic signal that results from the thermoelasticexpansion. Block 12. The photoacoustic signal may be a wave or othersignal. Detection may include, for example, using a suitable detector todetect an acoustic wave that travels through the test sample. Oneexample includes detecting the deflection of a diaphragm in fluidcontact with the solution when the acoustic wave contacts a sensordiaphragm. Another is using an optical detector to measure a signal suchas a perturbation (which may be a refraction change) in the samplefollowing the thermoelastic expansion.

By way of further example, FIG. 2 presents an additional example methodof the invention. This example embodiment is directed to a method fordetecting a pathogen, such as a cancer tumor cell, in a blood sample.The particular analyte application for this example embodiment couldeasily be changed, however.

In an initial step, a blood sample is obtained from a patient. Block 20.The sample may be, for example, as small in volume as a fraction of amilliliter to a quart or more. It may be obtained through a needle prickof the skin, intravenously, or the like. The white blood cells are thenseparated from the blood sample. Block 22. This may be done, forexample, using a centrifuge or other known separation method. The amountof white blood cells separated will depend, at least to an extent, onthe size of the initial blood sample.

The thus separated white blood cells are then placed in a carrier fluid.This step may include, for example, suspension in a saline solution.Block 24. Suspension, as used herein, is intended to be broadlyinterpreted as meaning diluted with, carried with, placed in, or thelike. Suspension of white cells in a carrier liquid, for example, mayinclude diluting the cells in the liquid, mixing the cells into theliquid, or the like. The white blood cells may include one or morecancer tumor cells, such as melanin.

In an optional step of this example method, a plurality of targetparticles is adhered to the cancer cells. Block 26. The target particlesor spheres can have a diameter of less than 0.01 millimeter. Theparticles may be, for example, micro-scale synthetic spheres selectedfor their ability to be detected by the electromagnetic energy that thesample will be exposed to. By way of particular example, goldnanoparticles, black latex spheres, dyes, quantum dots, or any othermolecule that will give the cell some color (such as biotin) may be usedthat are good absorbers of electromagnetic energy such as a laser light.

The target particles may be treated to cause them to be attracted to andadhere to the cancer cell. Treatment may include providing surfaceanti-bodies known to be attracted to the analyte of interest, electricalcharging, or the like. In one example step, the targets are attached bycreating a bond via an antibody-receptor pair. This provides thespecificity and strength necessary for the attachment. The targetparticle is functionalized to attach to the specific antibody. Thesefunctionalized particles are introduced to the sample containing thecells with the paired receptors. After washing away excess spheres thatdidn't attach to a cancer cell receptor, cancer cells have spheresattached to their receptors (usually surface receptors, though notnecessarily).

This optional step can be useful to “amplify” the detection threshold ofthe one or more cancer cells. An alternative step includes dyeing theanalyte with a suitable absorbing coloration. Excess target particles(that are not adhered to the cancer cells) can be removed from the testsample through washing or other known methods. Block 28.

The test sample is then communicated past a pulsed laser light. Block30. This step may include, for example, placing the sample in a systemof the invention that includes a reservoir, a flow generator such as apump, and a conduit connecting the reservoir to a test chamber where thelaser light is directed. When the cancer cell of interest is melanoma,for example, the laser light used to interrogate the fluid sample can beanywhere in the visible range, and in other wavelengths that melaninabsorbs. When the cancer cell is other than melanoma, detection will beachieved through absorption by the target spheres. The flowconfiguration may be designed to ensure that the sample is subjected tomultiple pulsed scans as it passes through the test chamber. As anexample, the fluid flow rate may be adjusted using the pump to causefluid to have a residence time in the test chamber wherein it issubjected to a desired number of pulsed scans, with examples includingat least 2, at least 10, at least 50 or at least 100. Multiple scans canbe useful to ensure accuracy, reduce risk that small concentrations ofanalyte escape detection, and for other reasons.

Also, methods and systems of the invention may include steps andelements for using different wavelengths of light to detect particularanalytes. If one analyte is known to absorb light of wavelength X andnot Y, for example, a test sample can be interrogated with both X and Ywavelength light. If a positive signal results when interrogated with Xbut not Y, an indication that the particular analyte is present isprovided.

The pulsed laser light is absorbed by the melanin and induces athermoelastic expansion in it, which results in an acoustic wave in thesolution. The acoustic wave is measured using a detector such as adiaphragm. Block 32. The magnitude of deflection of the diaphragm mayindicate, for example, not only the presence of a cancer cell butfurther an indication of its size, density, coloration, concentration,and other properties. Data from the diaphragm is recorded and displayedusing a controller. Block 34. This step may include, for example,amplifying an electrical signal from the diaphragm, recording it in amemory, and displaying it on a display.

Having now described some general example methods of the invention, moredetailed description of other methods, systems and details of inventionembodiments can be provided. In order to do so, some discussion ofphotoacoustics as it relates to some invention embodiments will behelpful.

Photoacoustics, also referred to as laser-induced ultrasound, uses shortduration pulsed light to create ultrasonic acoustic waves in anoptically absorbing medium. These acoustic waves are generated basedupon the thermoelastic properties of targeted analytes, which in manyinvention embodiment (but not all) will comprise chromophores.Chromophores by definition are atoms or molecules within a compound thatare responsible for the color of the given compound. Inherently, theypresent color by partially absorbing wavelengths composing the visiblespectrum of incident light. The non-absorbing wavelengths are subject toscattering and reflectance as determined by the composition of theparticular chromophore, thus emitting the color representative of thereflected wavelengths. Photoacoustics is based upon the absorbed portionof incident light. As light is absorbed by irradiated chromophores, theoptical energy gets converted into kinetic thermal energy trapped withinthe chromophore and subsequent thermal expansion of the atoms ensues.Thermoelastic expansion occurs when the condition of stress confinementis achieved by depositing direct energy unto a chromophore in acontinuous manner such that energy is unable to propagate away except bymeans of eventual convection with surrounding mediums. This condition isexpressed as:t _(p) <δ/c _(s)where t_(p) is the laser pulse duration, δ is the absorption depth oflaser energy, and C_(s) is the speed of sound in the medium. It isassumed that the absorption depth, δ is smaller than the diameter of thelaser beam.

Transient thermoelastic expansion can be achieved by irradiating withshort pulses of concentrated laser light allowing for elastic expansionand contraction of an absorbing molecule. This thermoelastic expansionand subsequent contraction results in the production of longitudinalultrasonic waves propagating in all directions away from the thermallyexcited medium of interest. Stronger heat generation will produce astronger acoustic wave via greater thermoelastic expansion. Therefore, astrong optical absorber emanates a strong acoustic wave. Conceptually,photoacoustics can be described as pulsed laser energy quickly absorbedby a scattering medium such that transient thermoelastic expansionresults in formation and propagation of acoustic energy. Thermoelasticexpansion, as used in photoacoustics, can be described by (assuming apure absorber where δ=1/μ₀)

${p(z)} = {\frac{1}{2}\mu_{a}{\Gamma\mathbb{e}}^{\mu_{a}z}}$where p(z) represents pressure at depth z, μ_(a) is the opticalabsorption coefficient of the tissue, Γ is the Gruneisen coefficient,which denotes the fraction of optical energy which is converted toacoustic energy. It is temperature dependent and is equal to 0.12 atroom temperature for most tissue.

The total photoacoustic energy resultant from the absorption of a beamis directly related to chromophore content. The amount of thermoelasticexpansion as given in the above equation is directly related to theabsorption coefficient μ_(a) of the chromophore. The absorptioncoefficient is derived from the molar extinction coefficient andchromophore concentration as described by: μ_(a)=2.3 εc where c is theconcentration of specific chromophore and ε is the molar extinctioncoefficient of the compound. These relationships are useful tocharacterize the chromophore above and beyond its detection. Forexample, the magnitude of the photoacoustic energy detected can beuseful to estimate the size, density, and identity of an unknownchromophore.

Acoustic pressure is proportional to energy per unit volume given asJoules per cm³. If a spot size is held constant, the integral ofpressure over depth yields the total absorbed energy as given by:E _(a)=∫₀ P _(o)(z)dzE_(a) is the total absorbed energy by the chromophore and P_(o)(z) isthe initial pressure as a function of depth z. The integral gives aquantity expressed in terms of J/cm², so the total energy detected isrelated to this quantity by the detector active area. The amount ofenergy (E_(a)) absorbed is directly related to the amount of incidentlight energy upon the medium (also given as J/cm²) to a certain limitdetermined by the absorption capacity of the specific chromophore.

Detection of these minute photoacoustic waves is equally as important astheir production in many invention embodiments. Photoacoustic detectionmethods can vary drastically from one experimental design to another.One element discovered to be useful in methods and apparatuses of theinvention is a piezoelectric copolymer in electrical connection with twoelectrodes, one being the ground wire and the other measuring thepositive voltage change of the film. These films may be constructed ofpolyvinylidene difluoride, or PVDF, and may or may not incorporate analuminum coating that acts as a conducting element. Many other detectorconfigurations are contemplated, however, including (by way of exampleand not limitation) other diaphragms whose deflection may be measuredwhen an acoustic wave strikes it, optical detectors that detect a changein reflective and/or refractive index in a fluid as an acoustic wavetravels therein, and the like.

As longitudinal acoustic waves propagate towards the piezoelectric filmof one example system and method, pressure accumulates according to themagnitude of each pressure wave. As these pressure waves come intocontact with the piezoelectric film, the lateral surface of the filmimpacted by the pressure wave shifts, disrupting the entropicallystabilized bilayer. The disruption of the polymer layer causes anelectrical charge to form between the two copolymer layers that can bedetected by two conducting electrodes as a voltage spike. Usinginformation on the magnitude of the signal (e.g., the amplitude of thevoltage spike) and/or the time difference between sending a laser pulseand receiving the pressure wave, chromophore density and concentrationcan be quantified as well as relative location to that of the PVDF filmas described in detail below. Some systems and methods of the inventioninclude elements and steps of taking these measurements and using theresultant data to measure density and the like.

As discussed above, some examples of the present invention includeapparatuses and methods for detecting the presence of melanotic cancercells within the human hematogenic system. Some systems and methodsemploy photoacoustic technology as an in vitro method of screening andquantifying disseminating tumor cells for the purpose of cancerdetection or as a method for determining the effectiveness ofchemotherapeutics. Such devices and methods offer a way of improvingupon standard detection protocols while alleviating many of theproblematic symptoms of alternative detection methods of the prior art.

Some photoacoustic detection systems and methods of the inventionprovide benefits and advantages including the ability to accuratelypinpoint both early and late stage disseminated cancerous cells presentwithin the bloodstream. This provides a relatively pain-free and lowercost alternative to clinical protocols of surgically based detectionmethods as well as offer a much needed mechanism for the early detectionof disease. It is proposed that this detection system may provide amethod for precise and unprecedented detection thresholds by identifyingsingle melanoma cells in the presence of millions of secondary bloodconstituent cells.

One example detection device provides a flow system through whichsolutions of interest may be introduced for the purpose of exciting asfew as a single melanotic cancer cell by pulsed laser excitation. Inorder to do so, a transparent flow cell, or excitation chamber,conducive to laser excitation is incorporated into the system. Areliable acoustic detection system or similar detection element is alsoincluded to capture photoacoustic waves. A signal element is used toconvert acoustic waves produced by melanotic excitation to voltagesignals that can be displayed for analysis.

An example detection system 50 of the invention is schematically shownin FIG. 3. It includes, among other elements, an excitation chamber orflow cell 52. The flow cell 52 is the chamber where laser excitation andacoustic wave propagation and detection occurs. A number of differentflow cells are suitable for use in methods and systems of the invention.Some include transparent sidewalls that allow an electromagnetic energysource, with an example being a laser, to be located external to thecell. One example cell 52 found to be useful is a customized flow cellcommercially available from Spectrocell, Oreland, Pa.). The cell 52 isshown schematically in FIG. 3, and is illustrated in greater detail inFIG. 4.

FIGS. 4(A) and 4(B) show the flow cell 52. It has a generallyrectangular box three dimensional shape, including opposing first andsecond narrow sidewalls 54 joined to wider opposing sidewalls 56.Dimensions shown are illustrative only, other dimensions will be useful.As shown by FIG. 4B, in the example cell 52 the narrow sidewalls have awidth of about 2 mm (along the width of FIG. 4B) and the wider sidewallshave a width of about 10 mm. The cell 52 also includes a detectiondiaphragm 58 arranged on one of the wider sidewalls 56 and covering acylindrical passage of about the same diameter in the sidewall 56whereby it is in fluid contact with the test sample in the cell 52interior. In the example cell 52, the diaphragm 58 is a 5 mm diameterPVDF film. The cell 52 also includes a positive electrode 60 extendingout from the cell interior and through a small passage in one of thenarrow sidewalls 54 and in fluid contact with the test sample therein.

FIG. 4(B) is a schematic showing dimensions of the example flow cell 52and an incident laser beam 64 direction as it passes through the cell 52from an external source. The example flow cell 52 has dimensions ofabout 2×10×45 mm (walls 54 width×wall 56 width×height) for a totalfluidic volume of about 0.9 ml. Other dimensions are contemplated andwill be useful. Particular dimensions will be selected based on designconsiderations including energy beam size, desired test samplevolumetric throughput, flow rate, and the like.

The top and bottom of the flow chamber 52 are tapered to cylindricalports 66 with an inner diameter of about 2.7 mm and an outer diameter ofabout 4.95 mm. These ports 66 serve to connect the cell to the conduit68 (FIG. 3) that communicates the test sample fluid into and out of thecell 52.

The high intensity laser beam 64 is directed into one of the narrowsidewalls 54 of the flow cell 2, opposite that of the detectiondiaphragm 58, at a height approximately equal to that of the detectionaperture 54. The laser beam 64 passes through the fluid test sample inthe cell 52 interior and out through the opposing narrow sidewall 54.

As discussed above, the laser beam is configured to induce athermoelastic reaction in the test sample which results in an acousticwave. In some methods and apparatuses of the invention, the acousticwave is detected through use of a diaphragm 58 that deflects when theacoustic wave contacts it. The example diaphragm 58 are in fluid contactwith the test sample. Deflection of the diaphragm 58 may be detectedthrough any of several methods. In the example flow cell 52, thepiezoelectric film diaphragm 58 on the wider cell sidewall 56 isemployed as an acoustic wave sensor diaphragm.

As is best illustrated by the schematic of FIG. 5, the diaphragm 58 isarranged about a pair of electrodes including the internal electrode 60and an external electrode shown generally at 80 which will be describedbelow. This configuration results in an electric field between theelectrodes 60 and 80 to be disturbed and fluctuate when the diaphragm 58is deflected therebetween. As will be discussed in detail herein below,the electric field fluctuation can be measured and used to signal thepresence of an acoustic wave, and therefore an anlayte. The amount ofdeflection of the diaphragm can be used to estimate qualities of thedetected analyte, including for example its coloration, density, massand/or size.

The diaphragm or piezoelectric film 58 in the example system 50 is cutto a diameter slightly larger than a 5 mm diameter passage on one of thewider (10 mm) sidewalls 56 and is affixed to the outside of the flowcell 52 by a 100% silicone sealant available from DAP Inc., Baltimore,Md. The piezoelectric film 54 is a 100 micron polyvinylidene difluoride(PVDF) copolymer film available from Ktech Corp., Albuquerque, N. Mex.

Although many other configurations are contemplated, copper electrodesare used for both positive signal detection and grounding. Differentmechanisms were used for each. The example positive electrode 60 is astripped 0.6 mm diameter copper wire approximately 17 mm in lengthinserted into the narrow (2 mm wide) sidewall 54 of the flow cell 52 asshown in FIGS. 4A-B and FIG. 5. The electrode 60 is sealed with 100%silicon sealant. It extends over much of the horizontal 10 mm width ofthe wider sidewall 56 and leaves an external end which is connected by aconductor such as wire 82 for transfer to a controller, which mayinclude one or more of a computer, an amplifier 84 (FIG. 3),oscilloscope 86 (FIG. 3), or the like.

The negative electrode 80, or ground, consists of a thin, circular 4 mmdiameter copper plate 90 from Small Parts, Inc., Miami Lakes, Fla.,soldered to a short length of 3.5 mm diameter wire which is in turnsoldered to a 31-221-VP BNC connector 92 from Jameco Electronics,Belmont, Calif. which connects to a grounded RG 58 coaxial cable 94 fromPomona Electronics, Everett, Wash. The flat copper plate 90 is flushagainst the external side of the PVDF film 54. This provides a ground toone side of the piezoelectric film.

Referring once again to the schematic of FIG. 3, experimental testsample solutions are pumped through the circulation conduit 68 using apump 102. In the example system 50, the pump 102 is a peristaltic pump,namely a Masterflex L/S Economy Drive.

It is noted that although many systems and methods of the inventioninclude circulation of test samples, others do not. Circulation isbeneficial in many applications, however, since it allows for arelatively large sample to be subjected to a small, compact energy beamas it is circulated past the beam.

The circulation conduit 68 in the example system comprises L/S 14platinum-cured silicon tubing available from Cole-Parmer Instruments,Vernon Hills, Ill. Although other pumps can be used, a peristaltic pumpoffers advantages in that no mechanical pump elements directly interactwith the fluid sample in the conduit 100. In the example system 50,about 15 ml of test sample solutions are entered into a 13.75 cm³ openreservoir 104. The peristaltic pump 102 provides negative pressure,circulating test sample fluid out of the reservoir 102, urging itthrough the test cell 52, and finally returning it to the reservoir 104through an open top. The silicon tubing 68 is size fitted to the outputand input ports 66 (FIG. 4) of the flow cell 52. In the example system50, test sample solutions circulate at a speed averaging about 9ml/minute, allowing for analytes of interest to be excited 235 timeswhen vertically crossing the beam path 64 (FIG. 4) of a 5 ns pulsedlaser with a 1 mm spot size (average conditions for given setup).

Those knowledgeable in the art will appreciate that a variety ofdifferent electromagnetic energy sources will be useful with methods andapparatuses of the invention. Lasers, and particularly pulsed laserlight, are one example believed to be useful. A wide variety of lasersare suitable for use in different methods and systems of the invention.Factors such as light wavelength, beam width, intensity, and the likecan be specified as desired for a particular application.

In the example system 50, photoacoustic excitation is made possible by asharply focused pulsed laser system shown generally at 110 in FIG. 3.Although a variety of different lasers and other electromagnetic energysources can be used, the laser system 110 in the example system 50includes a Q-switched, frequency tripled Neodymium-doped YttriumAluminum Garnet or Nd:YAG laser from Quantel Les Ulis, Cedex, Francehoused in a Vibrant Integrated Tunable Laser System from Opotek,Carlsbad, Calif. The Nd:YAG is a pumped laser that emits 1024 nm pulsedlaser light by an optical switch dubbed the “Q-switch” which acts as agate to release light at the maximum neodymium ion inversion. Oncereleased, laser light is reflected by two mirrors within the Vibrantinto a second harmonic generator where the wavelength is converted to532 nm then into a third harmonic generator where it is converted to 355nm. The 355 nm laser light is pumped into an Optical ParametricOscillator or OPO.

The OPO contains a Beta Barium Borate crystal that tunes by theelectronically controlled rotating of the crystal with respect to beam.The OPO converts the 355 nm input wavelength into two beams, the signaland the idler, each having longer wavelengths than the input beam. Acavity termed the Double Resonating Oscillator oscillates both the idlerand the signal wavelengths while the tuning angle of the crystaldetermines a phase match that produces the desired wavelength which isthen released from the cavity. The tuning angle is controlled by astepping motor operated by an external, computer generated program.

The OPO allows for the output of variable wavelengths ranging from 410nm to 710 nm. The particular wavelength of light utilized in methods andsystems of the invention using a laser will depend on applicationsincluding the wavelength that will be absorbed by the analyte beingsearched for, and the like. By way of example, laser light of about 450nm can be useful.

Following the OPO, the beam passes through a polarizer. In some systemsof the invention, the laser light is then coupled by a lens into a fiberoptics fiber carrier (with an example being a 1.5 mm standard silicafiber) for light transfer. While this can be useful, in someapplications the fiber can be subject to breaks and loss of energyresulting in poor results. Other example devices and methods, includingthe example system 50 utilizes an “open” delivery system, wherein laserlight travels through the atmosphere from the laser system 110 to thetest cell 52. In the example system 50, use of open light increasedenergy input into the test cell 52 from a range of 7.0-8.0 mJ outputwhen using fiber to 11-12 mJ.

In some example systems and methods of the invention, a tradeoff canexist between radiant energy strength and sufficient radiant exposure.That is, a radiant beam is desirably large enough to encompass all theanalyte or chromophores that may pass through the detection cell 52 butnot be too large such that it will not provide enough energy forproficient signal strength. This tradeoff must further be balancedagainst costs of a laser system 110—while conceivably a large enoughsystem could be obtained to provide an extremely large diameter beam ofextremely large energy, costs for doing so may be prohibitive.

The laser beam 64 exiting the output port of the example Vibrant lasersystem 110 takes the form of an ellipse. In such example methods andsystems, the beam should be focused into the flow cell 52 so that thebeam profile takes on a circular shape ranging between about 1 mm and 2mm in diameter so as to cleanly enter the 2 mm wall of the flow cell 52.Different size beams will be useful for different size cells, butgenerally it is advantageous to configure the beam to have a diameterthat extends across the entire width of the cell 52. This allows for theentire cross section of the circulating test sample to be irradiated bythe beam. Further, configuring the test cell in a geometry such as thatshown in FIG. 4 wherein the rectangular box shaped cell has a thin side(e.g., the 2 mm side) and a wider side (e.g., the 10 mm side) can beadvantageous. In such geometries, the beam 64 can pass through opposingnarrow sidewalls 54 and irradiate the entire cross section of thecirculating sample within the test cell 52.

Referring again to the schematic of FIG. 1, in the example system 50 theoutput beam 64 was spatially oriented by a series of lens showngenerally at 112. These include a cylindrical lens 114 (lens no.LJ1014L2-B, from Thorlabs, Newton, N.J.) in addition to two collimatingplano-convex lenses, 100 mm (element 116) and 50 mm focal length(element 118) (lens nos. LA1509 and LA1131, also from Thorlabs, Newton,N.J.) before entering the flow cell 52. The result was the beam 64having a 1 to 2 mm diameter cylindrical spot size measuring between11.0-12.0 mJ when it enters the cell 52. Spot size was determined byirradiating ZP-IT Laser Alignment Paper from Kentek Corp., Pittsfield,N.H., with a single 5 ns pulse and measuring the diameter of the burn.

In order to more fully illustrate the excitation and detectionmechanisms of some systems and methods of the invention, the schematicof FIG. 6 is presented. The photoacoustic mechanism as it pertains tosome example methods and systems of the invention begins with theexcitation of an analyte or chromophore 150, in this example casemelanoma or its related tissue phantom. In the example system 50 andrelated example methods, laser light 54 is pulsed at a rate of about 5ns and is at a wavelength of about 450 nm. The frequency of the pulsesmay be selected as desired depending on factors such as cell geometryand scale, sample flow rate, analyte being searched for, and the like.In many example systems and methods, frequencies of faster than amillisecond are believed useful, faster than a microsecond, and in manyapplications (such as the example system 50) on the order of nanosecond(ns).

This bombards the chromophore 150 of interest in the test sample as itpasses through the beam 64. The chromophore 150 absorbs at least aportion of the incident light from the beam 64 and photon energy istransformed into thermal energy. The increase in thermal energy causes atemperature rise within the chromophore 150 and a kinetic thermo-elasticexpansion ensues. The thermo-elastic expansion is transient due to thepulsed nature of the incident laser light 64. Since the excitationpulses are short enough in duration that no thermal heat escapes thechromophore 150, small elastic enlargements of the cells 150 occurwithin the solution in a manner such that pressure waves 152 arepropagated away from the source in the range of 1-50 MHz.

Longitudinal pressure waves, such as those emanated from a photoacousticsource, propagate parallel to the direction of the wave. The resultingeffect is that of a compression wave or moving band of high pressure.This high pressure band moves freely in solution (such as the testsample environment within the flow cell 52) away from the source in alldirections. The speed of the wave varies with application, and may befor example, at about 5 mm per second. A portion of these waves 152 comeinto contact with the diaphragm 58 and strike it as a mallet wouldstrike the surface of a drum.

The PVDF film 58 is a highly ordered copolymer that can be likened to alipid bilayer in a biological system. At steady state, or ground,conditions are entropically favorable for an ordered copolymerstructure. When an acoustic wave 152 strikes a surface of the film 58,that copolymer layer is disrupted, increasing the entropy of the systemand consequently forming a charge on the disrupted surface. The exteriorof the film 58 is grounded by the copper plate electrode 90 (FIG. 5)therefore conferring a positive charge to the interior surface of thePVDF film 58.

In other example embodiments, the PVDF film or other sensor may bedecreased in size which may increase signal strength by decreasing thesurface area of the detector and eliminate some signal dampening.Chemicals such as Indium Tin Oxide may be applied to the positivesurface of the PVDF to increase conductance at the surface of the film.

The positive voltage produced by the photoacoustic phenomenon isconducted throughout the interior of the test cell 52 by a conductivecarrier solution such as the 1.8% saline solution which is used in someexample methods and systems of the invention. The electrode 60 describedabove carries the charge out of the flow cell 52 for further analysis.

In the example system 50, the resulting electrical signal is amplifiedfor ease of detection. The transduced voltage of a photoacoustic effectis carried to amplifier 84 (FIG. 3) which is in the example system is aSR445A 350 MHz Amplifier from Stanford Research Systems, Sunnyvale,Calif. where it is amplified by four stages each providing a gain of 5for an amplification range of 5 to 125. In the example system 50,voltage signals are displayed by the oscilloscope 86, which in theexample system 50 is a TDS 2024 200 Mhz Oscilloscope from Tektronix,Beaverton, Oreg., triggered by a photodiode available from Thorlabs,Newton, N.J., upon each laser firing.

It is noted that the amplifier 84 and oscilloscope 86 are one example ofelectronics useful to receive and analyze signals from the test cell 52.In other example systems, these components are replaced by a singlecontroller, which may be, for example, a processor based device withinternal components useful to amplify and process the voltage signalfrom the cell 52. Such a controller may output data including visualand/or audio data indicating presence of an acoustic wave and thereforea chromophore of interest. The controller may also record data in amemory, and record other data such as time, test sample identificationand the like. The controller may be, for example, a computer.

As noted above, FIGS. 3-6 are schematic only, and have been provided toillustrate various elements of one example embodiment of a system andmethod of the invention. Other systems and methods will vary from thoseillustrated above. The system as illustrated, in fact, represents anexperimental apparatus as may be configured in a laboratory, forexample. Commercial embodiments of systems and methods of the inventionmay be configured using alternate conduits, cells, reservoirs, and thelike.

By way of example, some commercial embodiments may be configured in ahousing, using a computer controller that is linked to the laser, pump,and that provides data acquisition, recording, processing and display.Some embodiments of the invention may value portability and beconfigured as such. Other variations will be apparent to thoseknowledgeable in the art.

By way of example, an additional system of the invention was configuredusing an alternate cell configuration. The cell was configured as a dualchamber device constructed from standard microscope slides availablefrom Fisher Scientific, Pittsburgh, Pa., cyanoacrylate from Loctite,Avon, Ohio, 100% silicone sealant from Dow Corning, Baltimore, Md., andNylon sheet plastic from Small Parts Inc., Miami Lakes, Fla. Thesematerials were carefully cut and constructed by hand and sometimesresulted in results that were less reliable than those obtained usingthe example system 50, although the configuration will be useful forsome applications. One chamber served as the flow chamber and conducteda positive signal, similar to the flow cell 52 of FIGS. 3-4. The secondchamber was a stagnant reservoir filled with saline solution that wasgrounded by an electrode inserted into the chamber.

Separating the two chambers was a glass divider with a 5 mm diameterpassage cut into it. The PVDF film was sealed onto the face of thedivider on the grounded side. Consequently, this mechanism functioned inthe same manner as the diaphragm in the example system 50 and was usedto realize the idea that a proposed photoacoustic device. It wasessentially consistent with the cell 52 discussed above, except that thesecond saline filled chamber replaced the grounded electrode 80.

Other variations of the cell 52 have likewise been utilized. By way ofone example, in an alternate configuration the internal electrode 60 waseliminated. This configuration utilized an additional positive, copperplate electrode housed on the opposite side of ground electrode 80 thatclamped both sides of an aluminum plated PVDF film together. This filmreplaced the diaphragm 58 shown above. The aluminum plating can serve asconductors for both the positive and ground signals, avoiding the use ofthe invasive electrode 60 into the flow cell.

It was discovered, however, that the aluminum plating to some degreecompromised the structural integrity of the PVDF film 58 and resulted inpoor signal strength. It is believed the lack of signal strength mayhave been a result of signal shorting by electrode contact. To addressthis issue the example system 50 shown above utilizes the non-platedfilm 58, internal positive electrode 60, and direct grounding by anexternal plated electrode 80 placed directly on the detection area ofthe PVDF film 58. Although the configuration shown above provedbeneficial over the plated film configuration in some applications, thisalternate electrode configuration may be useful in some applications.

Other variations and modifications of the system and method as shownabove are also contemplated. Some modifications, for example, will beuseful to adjust the threshold of detection of systems and methods ofthe invention. This can be adjusted, at least to a degree, throughadjustment of signal strength. Increased signal strength will generallyresult in increased sensitivity. Increases in signal strength, however,must be balanced against resultant signal noise. A high signal to noiseratio is desirable. Those knowledgeable in the art will appreciate thattuning of a system to achieve this will be possible and may vary onparticular application details. Some example system and method elementsthat can be varied to affect signal strength and noise ratio arediscussed below.

For example, an improvement in signal strength in the example system 50was achieved due to an increase in detection aperture size in thesidewall 56 underlying the diaphragm 58 from about 2 mm to about 5 mm indiameter. Generally, larger detection apertures increase the likelihoodthat acoustic wave collection will occur from the surrounding medium. Anadditional improvement in signal strength was achieved though using arelatively small overall cell volume, and particularly in regards to thenarrow 2 mm side wall 54 width. This narrow 2 mm sidewall 54 widthconstrains the chromophores passing through the flow cell and forces theentire medium to pass through the excitation beam path assuring that nochromophore passing through the cell is missed.

Although the 2 mm value is only one example of a useful size, manymethods and systems will benefit by using a first sidewall 54 that isnarrower than a second sidewall 56, with the electromagnetic energysource directed through the first narrow sidewall 54. When the beam hasa width that extends over across substantially all of the narrowsidewall 54, the entire cross section of the circulating test sample isirradiated. Directing the beam through the wider sidewall 56, on theother hand, would require a significantly wider beam (with resultantsignificantly lower per unit area energy).

Other configurations are possible to achieve this result, including, forexample, test cell configurations with a narrow “throat” portion throughwhich the test sample flows at a high velocity. The velocity should notbe so great, however, as to shorten the residence time of analytes inthe energy beam. One example configuration is discussed herein below.

It is also noted that there are benefits to locating the detectiondiaphragm 58 on the wider sidewall 56 through which the beam 64 does notpass. This wider sidewall 56 provides a larger area so that largerdiaphragms 58 can be used. Also, in some experiments it was discoveredthat the beam 64 could cause increased noise in the detection signal bya pyro-electric effect of light photons interacting with the polymers ofthe film. Better performance was achieved when the beam was directedthrough the narrow sidewall 54 and direct interaction with the diaphragm58 was avoided. In some example systems and methods, however, frontexcitation may be useful.

In other embodiments of methods and systems of the invention, anothermodification has been provided to further improve performance. Elementsfor adjusting the location of the laser beam 64 and the cell 52 relativeto one another may be provided, for example. Although many possiblemechanisms are possible to achieve this, an x-z translational stageconnected to one or both of the laser 110 or cell 52 is useful. This canbe useful to ensure the beam accurately encounters the test cell 52.Such a mechanism has been schematically illustrated as X-Y-Z adjuster170. In practice this may comprise a controllable table underlying thelaser 110 or cell 52 or the like.

Other potential modifications of the system 50 include using a fiber tocommunicate laser light as described above in order to maneuver thelight source to the most efficient location for excitation of the cell52. Trial runs were conducted using light emitted directly from a fiberand were found to result in a spot size of nearly 4 mm. It has beendiscovered that the focus of the laser light was important and directlyrelated to signal strength. It has also been discovered that a smalldecrease in spot size diameter resulted in an increase in radiantexposure by the inverse square law. As discussed below, use of fiber wasfound, at least in the example system 50, to decrease signal intensity.

Radiant exposure is defined as Joules per cm². As it pertains to thedesign of some systems of the invention, a millimeter decrease in spotsize from 3 mm to 2 mm for a 12 mJ light source results in a 125%increase in radiant exposure. Therefore, a 35 mm plano-convex lens (lensno. LA1027 from Thorlabs, Newton, N.J.) was inserted between the fiberand the detection chamber to decrease the incident spot size toapproximately 2 mm in diameter. Eventually, the entire setup wasrepositioned for excitation without an intermediate fiber, as describedabove with reference to the laser system 110 and its lens 112. Thisincreased incident energy from 7.0 or 8.0 mJ to 11.0 or 12.0 mJ. Thespot size was brought to within 0.026 cm³ (between 1 and 2 mm indiameter) giving a radiant exposure in the range of 460-900 mJ/cm² andincreasing signal voltages to their highest level.

The amplification of the signal as it passes from the detectionapparatus to the oscilloscope also can play an important role in theidentification of a signal as well as noise response. In the examplesystem 50, it was discovered that a gain of 25 appeared to produce thebest signal differentiation. A gain of 125 often dropped the signal tonoise ratio significantly and produced poor results, although it shouldbe noted that in some instances a gain of 125 was preferable for thedetection of extremely weak signals. Therefore, in the example system50, a gain of 25 is used as a default, however, the gain should be seton the basis of the individual experiment or application.

Detection trials were run using the example system 50 and methodsdiscussed above. Latex microspheres were used as a precursor to livemelanoma cells. The test solutions were also used to determine adetection threshold for the example system and method in order tocharacterize the sensitivity of the example system. Some experimentalruns were conducted using a dual chamber design while initialthresholding was performed using a single chamber design for the flowcell.

Black CML Latex Microspheres (no. 2-BK-7000 available from InterfacialDynamics Corp., Portland, Oreg.) measuring 6.6 μm in diameter were usedas tissue phantoms to mimic the photoacoustic response of melanoma cells(optical properties of melanoma cells and tissue phantoms are describedbelow). The latex microspheres were chosen because of their broadbandabsorption spectrum and relative size. A standard melanoma cell canrange between 10 μm to greater than 50 μm depending upon theheterogeneity of the cell line and morphology of the cell. Although themelanoma cell tends to be much larger than the microspheres used inthese trials, the microshperes contain a darker pigment and thereforesufficiently imitate the photoacoustic effect of a melanoma cell, asdiscussed below. However, it is speculated that the larger size of themelanoma cells and the large number of melanin granules contained withinthem will allow them to produce a stronger photoacoustic signal than themicrospheres per single cell. Therefore, it is believed that truemelanoma will provide a signal similar to that of the microspheres, ifnot greater.

Spectroscopic analysis of the latex microspheres was conducted using aHR-2000 High-Resolution Spectrometer and a HL-2000 Halogen Light source(from Ocean Optics Inc., Dunedin, Fla.). An absorption spectrum of 4.3g/100 ml (4.3%) black microspheres was taken using a 150 μm cuvetteconstructed of 1 mm thick glass slides (from Fisher Scientific,Pittsburgh, Pa.) and 150 μm shim stock (from Artus Corp., Englewood,N.J.). The microsphere solution was placed within the constructedcuvette and inserted between the halogen light source and detector ofthe spectrometer. Data was analyzed using OOIBase32 (from Ocean OpticsInc., Dunedin, Fla.). Data collection was taken under an integrationtime of 14 ms with no averaging. A short path length was used reduce theeffects scattering on the absorbance data. The resultant absorptionspectrum confirms that the microspheres possess steady absorption acrossthe entire visible spectrum showing that the spheres are indeed a blackbroadband absorber. This spectrum indicates a slight increase inabsorbency with wavelength within the visible range.

The latex microspheres were introduced into a signal conducting carrierfluid. In the experimental runs made, saline solution was used. Othercarrier fluids will also be useful. Initially, the saline solution was a0.9% sodium chloride (NaCl) solution created by adding 0.9 g of solidNaCl solute into 100 ml of de-ionized water solvent. In the experimentalruns made, a 1.8% solution was used, although many other concentrationswill be useful. Microspheres were added to 20 ml of saline solution invarying concentrations ranging from a maximum of 7.124×10⁶microspheres/ml to a minimum of 7.0×10² microspheres/ml. The microsphereconcentration of each solution was deduced by:

${{microsphere}/{ml}} = {( {\frac{V_{s}}{V_{m}}/V_{su}} ){( V_{i} )/( {20\mspace{14mu}{ml}} )}}$where V_(s) represents solid volume of microspheres from factory, V_(m)volume of a single 6.6 μm microsphere, V_(su) the remaining volume offactory microsphere suspension, and V; the volume incorporated intosaline solution. 15 ml of each selected concentration was placed intothe reservoir for circulation through the detection system.

Various concentrations of spheres were run using systems and methods ofthe invention. Trials employed a 0.9% saline solution, 510 nmexcitation, 7.5 mJ input energy, and a 2.36 mm×2.45 mm spot sizeresulting in a radiant exposure of 0.129 J/cm². The signal was averagedover 64 acquisitions. Results of these tests confirm that systems andmethods of the invention are not only useful to detect the microspheres,but further are useful to estimate concentration of the microshperes.

Two additional sets of experiments were conducted to determine thesensitivity of one example system and method. In the first, sampleconcentrations ranging from 7.12×10⁶ to 7.0×10² microshperes/ml wereused to gain an understanding of signal variation with concentration.Determining this makes it possible to quantify unknown concentrations oftissue phantoms by known signal intensities. This represents animportant benefit of some systems and methods of the invention—signalintensity can be used to estimate qualities such as analyte density,analyte concentration, analyte size, and other properties.

All trials in the first set of experiments were run with the followingexperimental parameters: 450 nm excitation, 8.75 mJ energy input, 1×1 mmspot size, 13.1 mm beam length, radiant exposure of 0.875 J/cm², and 9ml/min flow rate. Samples 7.12×10⁶ through 8.9×10⁴ employed a signalgain of 25 averaged over 64 acquisitions. Lower concentrations rangingfrom 5.5×10³ through 7.0×10² used a signal gain of 125 averaged over 128data acquisitions. The differing parameters were useful to differentiatesignals of lower concentrations for this setup. The experimental resultsof these experiments again confirmed that photoacoustic detection ofcirculating cells can be achieved using systems and methods of theinvention as described herein. Latex microspheres, 6.6 μm in diameter,were successfully detected at 3 concentrations on the order of 10⁶ permilliliter.

In the second set of experiments seven microsphere solutions ofdifferent concentrations ranging from 8.9×10⁴ microsphere/ml to 7.0×10²microsphere/ml were passed through a system of the invention. Trialswere run with the following parameters: 450 nm excitation, ×25amplification, averaged over 128 data acquisitions, flow rate of 9ml/min, laser energy input 11.5-12.0 mJ, spot size of 0.13×0.2 cm (0.026cm²), and a radiant exposure of 0.461 mJ/cm².

These experiments suggest that variations in signal amplification gainmay be one example parameter modification useful to increasesensitivity. In the example system 50, gains of 25, 50 and 125 werefound useful. Higher gain settings resulting in greater detectionsensitivity. Too high of a gain setting, however, risks a high signal tonoise ratio.

In further consideration of sensitivity, assuming even distribution ofmicrospheres within the entire solution, the exact amount of singlemicrospheres crossing the beam path at any given time can be deduced by:M _(#)=(C _(m))(V _(b))Where M_(#) is the number of excited microspheres, C_(m) isconcentration of microspheres per milliliter solution, and V_(b) is thevolume of the excitation beam path through the flow cell in cm³. Thisdeduction makes it possible to realize the number of single cellsproducing each photoacoustic signal, therefore interpolating the numberof single chromophores necessary to induce a differentiated signal.

Also, during the experimental trials a more concentrated saline solutionwas used to boost signal strength by increasing conductivity throughoutthe test solution while decreasing resistance from water. Initially, aphysiological saline solution of 0.9% had been used as a solvent fortrials. A study was conducted that doubled the saline concentration to1.8% with a resultant increase in signal strength. Increasing the salineconcentration from 0.9% to 1.8% increased the peak signal of a solutionby a factor of 1.2. Although further increases in strength may beachieved with higher saline concentrations, at some level theconcentration becomes too high to support a live melanoma cell survive,since they begin to lyse at saline concentrations of above about 0.9%.It is believed that concentrations of about 1.8% provide a usefulmaximum, although in some circumstances higher concentrations may beutilized. Also, other solutions in addition to saline may be useful inpractice of methods and systems of the invention. Generally, conductingfluids that support a melanoma will be useful when using an acousticsensor that relies on an internal electrode. Other sensor configurationsmay allow for non-conducting fluids to be used.

Using the example system discussed above with a 1.8% saline solutioncarrier was shown to be very successful for the photoacoustic detectionof tissue phantoms in the form of 6.6 μm black latex microspheres. Theresults indicate that systems and methods of the invention are capableof detecting the presence of single cells in a circulating solution.

In order to further characterize some systems and methods of theinvention, further testing was performed using analytes other than or inaddition to the above described microspheres. An application that manysystems and methods of the invention will find particular utilityinclude (but are not limited to) detecting cancerous melanoma cellscirculating within the hematogenic system of a potential cancer patient.The example detection systems and methods of the invention can processmaterials through in vitro analysis which requires a method forextracting the cells of interest from a human patient. A simple blooddraw is the most common method for obtaining cells present in thecirculatory system and may be used as a relatively pain-free, routinemethod for obtaining particular samples of interest. Once a blood sampleis collected, it is proposed that metastatic melanoma cells can beaccurately isolated from whole blood in vitro by implementing, by way ofexample, a Ficoll-Hypaque centrifugation technique. This techniquerequires the extracted whole blood to undergo centrifuge gradientseparation in order to isolate the particular cell layer of interestbefore introducing it to the photoacoustic detection system.

To further evaluate systems and methods of the invention, tissuephantoms representing biological melanoma were introduced into healthysamples of blood in vitro and detected using the photoacoustic method.This section describes experimental details of such test runs. Beforeproviding such detail, however, some application background will behelpful.

Leukocytes, or white blood cells, defend the body against infectingorganisms and foreign agents, both in tissue and in the bloodstreamitself. Human blood contains about 5,000 to 10,000 leukocytes per cubicmillimeter; the number increases in the presence of infection.Leukocytes as well as erythrocytes are formed from stem cells in thebone marrow. They have nuclei and are classified into two groups:granulocytes and agranulocytes.

Granulocytes form in the bone marrow and account for about 70% of allwhite blood cells. Granulocytes include three types of cells:neutrophils, eosinophils, and basophils. Neutrophils constitute the vastmajority of granulocytes. The main purpose of these cells is to surroundand destroy bacteria and other foreign particles as well as act ininflammatory response mechanisms during infection or allergic reaction.Granulocytes serve as the first line of defense against infection byforeign cells.

Agranulocytes include monocytes and lymphocytes. Monocytes are derivedfrom the phagocytic cells that line many vascular and lymph channels,called the reticuloendothelial system. Monocytes ordinarily number 4% to8% of the white cells. They move to areas of infection, where they aretransformed into macrophages, large phagocytic cells that trap anddestroy organisms left behind by the granulocytes and lymphocytes.Lymphocytes, under normal conditions, make up about 20% to 35% of allwhite cells, but proliferate rapidly in the face of infection. There aretwo basic types of lymphocytes: the B lymphocytes and the T lymphocytes.B lymphocytes tend to migrate into the connective tissue, where theydevelop into plasma cells that produce highly specific antibodiesagainst foreign antigens. Other B lymphocytes act as memory cells, readyfor subsequent infection by the same organism. Some T lymphocytes killinvading cells directly; others interact with other immune system cells,regulating the immune response.

Peripheral Blood Mononuclear Cells (PBMCs) is a term used to describemonocytes and lymphocytes that can be separated from a whole bloodsolution using a Ficoll-Hypaque centrifugation technique describedherein. Other separation techniques, including centrifuge and similartechniques, will also be useful with methods and systems of theinvention.

Although conflicting information exists on metastatic disease and itsinteractions with the human immune system, it is hypothesized thatantigens present on the surface of melanoma cells within the bloodstream of an individual with metastatic disease will be recognized andbound by these mononuclear cells. This assumption is based upon the ideathat metastatic disease is a chronic disease which persists in the bloodstream and would be primarily attacked by monocytes and lymphocytes thatare thought to defend against chronic illness more so than granulocytes.Therefore, isolation of the peripheral blood mononuclear cell layershould result in the isolation of any melanoma cells present in theblood stream.

Samples of healthy, cancer free blood were drawn from consentingindividuals from within the lab group. Samples were taken byvenipuncture from the antecubital area of the arm in the amount of 10-50cubic centimeters using a standard blood draw procedure.Ethylenediaminetetraacetic acid (EDTA) liquid coated tubes were used forblood collection to inhibit clotting. Blood samples were stored in arefrigerated environment for no more than five hours before beingprocessed.

A Ficoll-Hypaque separation technique was used to isolate the peripheralblood mononuclear cell layer from the whole blood samples. TheFicoll-Hypaque process employs a sugar compound of a specific densitythat separates specific blood components by a density gradient whencentrifugal force is applied. Approximately 1 ml of Histopaque 1077(from Sigma-Aldrich Inc., St. Louis, Mo.) separation gradient was placedin Pyrex No. 9800 glass tubes (from Corning Inc., Acton, Mass.).Approximately 7 ml of blood from the refrigerated samples were gentlypoured on top of the Histopaque 1077 and stopped with a rubber stopper.The sample tube was then placed in a 60 Hz 3400 rpm Vanguard 6500centrifuge (from Hamilton Bell Co., Montvale, N.J.) and spun for 10minutes. The resulting gradient and relative location includes theperipheral blood mononuclear cell layer consisting of monocytes andlymphocytes separated out directly above the Histopaque layer and belowthe plasma. The Granulocytes are larger and separate below theHistopaque layer, directly above the blood layer (not shown).

Following separation, the differentiated PBMC layer was carefullyremoved using standard transfer pipets (from Samco Scientific Corp., SanFernando, Calif.) and placed into 1.5 ml Flat Top Microcentrifuge Tubes(from Fisher Scientific, Pittsburgh, Pa.). The PBMCs in the microcentrifuge tubes were washed in a saline solution and re-spun for 5minutes. Excess saline solution and plasma was pipetted off of the topof PBMC layer. This was repeated until peripheral blood mononuclearcells were cleanly isolated.

Two suspensions of isolated mononuclear cells (0.111 g) were added to 20ml of a 0.9% saline solution. One suspension served ace; the control.The second suspension contained 0.5 ml (1.49×10⁸) of black latexmicrospheres. Both were introduced to an example photoacoustic detectionsystem of the invention.

A second test sample was created in which 1 ml (2.98×108) of 6.6 μmblack latex microspheres was added to 7 ml of human blood sample priorto FicollHypaque centrifugation. Peripheral blood mononuclear cell layerwas isolated as previously described, added to 20 ml of 0.9% saline, andintroduced into detection system.

All trials were run with the following parameters: 450 nm excitation,amplification×25, averaged over 128 data acquisitions, 9 ml/min flowrate, 9.5-10.5 mJ input energy, and a spot size of 0.16×0.16 cm (0.0256cm³) resulting in a radiant exposure of 0.39 j/cm². It is postulatedthat melanoma cells in the blood stream of a metastatic patient mayremain in the plasma when subjected to the Ficoll-Hypaque gradient. Forthis reason, a separate trial was conducted using isolated blood plasmato determine the dynamic ability of the example detection system tofunction using different mediums. Two samples were prepared, a controlsample consisting of 3 ml of human plasma and 10 ml of 0.9% saline and atest sample of the same solution with 0.5 ml (1.49×10⁸) blackmicrospheres added, to mimic the presence of melanoma in plasma. Similarexperimental parameters were used as before, only a 595 nm excitationwavelength was used to eliminate absorption by the naturally yellowpigmented plasma. After isolation of the peripheral blood mononuclearcell layer, a saline/mononuclear cell suspension was made to which1.49×10⁸ microspheres (7.12×10⁶ per ml) were added. Amplification wasset at 25, 450 nm excitation, and photoacoustic results for a controlagranulocyte cell suspension.

The resultant output control waveform for the mononuclear cellsuspension confirms that there is no photoacoustic excitation thatoccurs from exciting the PBMCs. This is expected since mononuclear cellsare white in color and should contain no active chromophores that mayproduce a photoacoustic signal. The photoacoustic waveform resultingfrom the addition of microspheres to an isolated mononuclear cellsuspension once again clearly confirms the ability of example systemsand methods of the invention being investigated to identify chromophoresamongst a circulating PBMC solution.

Also, the waveform resulting from the addition of microspheres to wholeblood previous to centrifugation is representative of melanoma cellspresent in the blood stream itself. The microspheres were added in largeconcentration so that if all the spheres were isolated properly,7.12×10⁶ microspheres/ml would result in solution. This was most likelynot the case as it can be assumed that many microspheres were lostduring the process of separation. The resulting waveform shows thatFicoll-Hypaque centrifugation is extremely successful in isolatingtissue phantoms representing melanoma cells, proving this hypothesiscorrect.

The extraction of blood plasma illustrates a second method for detectingchromophores amongst a non-saline solution. Microspheres were accuratelydetected at high concentrations at 595 nm. This shows the versatility ofwavelength modification for detection. 595 nm does not excite the yellowpigmented plasma (as seen in the control) primarily because plasmaabsorbs in the blue and red spectrum. Still, 595 nm providesphotoacoustic excitation for tissue phantoms present in solution due tothe broadband absorption of the microspheres. This provides a mode fordetection of cells isolated to the blood plasma, which will provebeneficial.

These experiments confirmed that photoacoustic response from tissuephantoms inoculated into healthy blood can be accurately isolated anddetected using photoacoustic detection systems and methods of theinvention illustrated above. Experimental results confirm that methodsand systems of the invention are useful to extract and identify cellsevenly distributed amidst hundreds of millions of blood constituentcells not of interest. It also shows the ability of the systems andmethods to express the presence of chromophores amongst a mononuclearcell suspension which eliminates the need for a further cell isolationmethod. It is believed that live malignant melanoma cells will reside inthe same FicollHypaque gradient as the tissue phantom cells. However, itis also possible that the melanoma cells, being larger than themicrospheres and of different makeup, may separate differently, possiblyto the granulocyte layer.

Detection trials described above have shown that example methods andsystems of the invention can successfully detect chromophores insolution on the order of twenty single cells or fewer. The sensitivityof one example system described above is as low as two microspheres.Other systems and methods of the invention can be modified throughselection of laser energy, beam area, test cell geometry, and similarparameters to achieve a detection threshold of as low as one cancercell.

An additional experimental evaluation of methods and systems of theinvention in applications directed to the detection of disseminatedcancer cells is to test the ability to detect melanoma cells in vitro.In order to do so, a live melanoma cell line was cultured so that anabundance of isolated melanin could be introduced into the system fordetection. The below discussion describes the cell line used and cellculturing methods employed, and describes in detail methods and systemsof the invention used for detecting the live melanoma.

Some background discussion on melanoma will be useful. Melanoma is amalignant tumor composed of unregulated melanocytes. A melanoma cell isessentially a melanocyte containing one or more mutations that inhibitnormal cell growth and regulation. A melanocyte produces coloring inmammals by secreting three unique pigments: the dark insoluble,nitrogenous eumelanins formed from the oxidative polymerization ofdihydroxyindolequinones, the sulphur-containing alkali-solublephenomelanins derived from cysteinyl-DOPA which provide the lightercolors (primarily brown and red-brown), and the amphoteric pheochromes(red and red-orange colors). These colors are enzymatically synthesizedby 10 nm granular sites studding the internal walls of the melanocytes.

These pigments are termed melanin and are produced in various shapes,sizes, and amounts depending upon the cell. The amount and type ofmelanin produced determines skin, hair, and eye color in mammals. Themelanin produced is encapsulated by the melanoma cell providing apigment for any cell producing melanin. It is the melanin granulesencapsulated within the melanocyte, or melanoma cell, that serves as thebroadband absorber to produce photoacoustic signals when exposed toincident laser light.

Melanins are a broad class of functional macromolecules that togetherexhibit a band structure model characteristic of an amorphous solid withbroad band absorption spectrum in the visible and UV. Eumelanin, theprevalent pigment, has been shown to efficiently absorb UV and visiblephoton energy and deactivate with a quantum efficiency of less than0.05%, which fits its role as a photoprotectant in skin. The mechanismby which this occurs is complicated and not entirely understood. Studiesshow that melanins consist of small heterogeneous oligomeric units thatpossess different redox states resulting in a broad range of HOMO-LUMOgaps (energy difference between the highest occupied molecular orbitaland the lowest unoccupied molecular or bital). This chemical disordermodel allows for broadband monotonic absorption as a consequence of thesuperposition of a large number of non-homogeneously broadened Gaussiantransitions associated with the components of the melanin ensemble.Despite all the work done to characterize melanin absorption it has beenhypothesized that the absorption spectrum of human melanin is dominatedby scattering, as it shows no characteristic absorption resonances inthe visible or ultraviolet.

Melanocytes are present in all epithelial based organs which give riseto cancerous tumors. It is therefore believed that melanoma cells arepresent in any epithelial based cancers, or carcinomas, and theirresulting metastatic cells. Unfortunately, approximately 10% of thesecarcinomas consist of amelanotic melanoma cells that do not producepigment therefore rendering the photoacoustic detection methodineffective. It is believed, however, that molecular tagging mechanisms,including dyeing and nanoparticle technology by way of example and notlimitation, could be used to identify and bind to amelanotic cells invitro which would allow for photoacoustic excitation of the boundmarkers. This could eventually allow for the photoacoustic detection ofany melanoma based cancer.

The Melanoma cell line utilized in experiments discussed below wasSK-MEL-1. These cells were originally obtained in 1968 from the thoracicduct lymph of a 29 year old Caucasian with rapidly progressing malignantmelanoma. The cells are known to be tumorigenic in nude mice orcortisone treated hamsters, producing pigmented malignant melanomas.Additional cellular characteristics included a spherical growthproperty, with cells loosely clustered exhibiting a granular cytoplasm.

The cells were grown in suspension in 25 cm² Canted Neck Flasks with aPhenolic Style Cap (from Corning Glass Works, Corning, N.Y.) at 37° C.in a humidified 5% CO₂ environment. Approximately 10 ml of the cellsuspension was kept in each flask. A media composed of 444.4 ml RPMI(from Invitrogen Corp., Grand Island, N.Y.), 5 ml Glutamine (also fromInvitrogen Corp.), 50 ml Fetal Bovin Serum (from U.S. Bio-TechnologiesInc., Pottstown, Pa.), and 0.6 ml Gentamycin (from AmericanPharmaceutical Partners, Schaumburg, Ill.) was used and renewed threetimes per week. When changing the media, 7 ml of the cell suspension wasremoved and discarded, leaving 3 ml in the original flask. Then, 7 ml offresh media was then added back to the flask.

The cells were counted before each media renewal. A small sample ofcells would be obtained for the count. The cells were gently mixed witha blue ink at a dilution of 1:2. Using a hemacytometer, the cells inthree squares were counted. A cytospin was performed on a selected batchof cells and a cell block was made and stained with Hematoxylin andEosin. Individual cells varied in size, with many containing dark,intracellular inclusions. The nuclei were centrally located, round, andcontained one or more prominent nucleoli.

The amount of melanin produced was found to vary by cell withapproximately 5% of the cells containing dense brown cytoplasmicgranules dispersed evenly throughout the cytoplasm. Therefore only 1 in20 live melanoma cells were actively producing melanin. Mutatedaneuploid state of the transformed melanoma is apparent through cellscontaining multiple nuclei.

A number of control trials were run using example methods and systems ofthe invention in order to definitively prove the detection of melanomacells as opposed to other absorbers or pyro-electric effects. Thesecontrol experiments were run in addition to detecting melanomasuspensions.

In addition to a plain 1.8% saline control solution (as prepared andtested in above discussion), a 7.124×10⁶ μs sphere/ml solution wasprepared using 6.6 μs White CML Latex Microspheres (from InterfacialDynamics Corp., Eugene, Oreg.). Whereas black microspheres representsthe absorbing effects of melanoma, white microspheres can be used torepresent the scattering effects of melanoma cells. Therefore, the whitemicrosphere suspension was introduced to determine the effects of apurely scattering medium on the system. Live melanoma cells act as bothabsorbers and scatterers due to their variable melanin content and largeamount of white surface area. It is possible that a scattering medium(such as melanoma) may produce phantom signals or oscillations due to apyro-electric effect, or signals produced by scattered photonsinteracting with the piezoelectric film or electrodes. These controltrials give examples of typical pyro-electric waveforms so as toeliminate the possibility of mistaking a pyro-electric signal for thatof a melanoma signal.

A second control experiment was conducted using a 1:1 suspension of RPMICulture Medium (discussed above) and 1.8% saline. The medium, used toculture the melanoma cells, contains phenol red that serves as anacid/base indicator to identify when the cells have expired thenutrients of the growth medium. Phenol red (Phenolsulfonephthalein) hasa pK value of 7.9. Different absorption spectra exist from differentsources and detection techniques as can be seen by comparing the twospectra. It is evident that phenol red has a variable absorbance at 450nm that could produce a photoacoustic signal but no absorbance at 620nm. Therefore, detection trials were ran at 450 nm and 620 nm for boththe culture medium suspension and the melanoma suspension forcomparison. This would ensure that any signal produced at 620 nm wouldbe that of the melanoma cells not that of the culture medium eitherpresent on the cells or absorbed by the cells. Thus eliminating thenotion that the culture medium may be producing a photoacoustic signalmistaken for melanoma.

Before suspending the melanoma for introduction into the detectionsystem, the cells were counted then spun at 1200 RPM at 4° C. for tenminutes using a Fisher Scientific accuSpin 3R centrifuge. Thesupranatant of media was then removed from the cell pellet formed. Next,the cells were washed using Dulbeccos Phosphate Buffered Saline(Invitrogen Corp., Grand Island, N.Y.). Saline was added to the pellet,the cells were gently mixed, becoming again suspended, and the procedurewas repeated using the same methods as described above. Finally, thesupranatant of saline was removed, and a small amount of fresh salinewas added to the cell pellet.

A 15 ml solution was made consisting of 2.3×10⁶ live melanoma cells and1.8% saline resulting in a 1.53×10⁵ cell/ml suspension. This suspensionwas entered into the example system of the invention and detected usingpulsed laser light with the following parameters: 450 nm and 620 nmexcitation, 9.5-11.6 mJ input energy, gain of 25, and averaging over 128data acquisitions.

The cell line chosen for these trials was a non-clustering metastaticmelanoma cell line. The original sample was taken from the metastatictumor of a class IV cancer patient which insinuates that the cellspossess an inherent ability to disseminate and intravasate into theblood stream. Therefore, the cells cultured and tested should becomparable to cells that would be present in the blood stream of ametastatic cancer patient. Approximately 5%, or 1 in 20, of the culturedcells produced visible melanin. The irradiated spot size for thesetrials was 0.13 cm×0.2 cm (0.026 cm2) with a beam length of 1 cm. If itis assumed that 1 in 20 cells produced viable melanin then it can bededuced that any resulting signal was produced by 200 melanotic melanomacells per irradiated beam path. 450 nm was used to excite the melanomasamples due to the optical absorption properties discussed above.

FIGS. 7A and 7B show resulting data, with FIG. 7A including results forthe live melanoma and FIG. 7B showing results for RPMI culture. Noisedata at the far left of each plot (near the origin) corresponds to noisefrom firing of the laser. As indicated by FIG. 7A, a distinct melanomasignal is evident at about 2.5 ms. No such peak occurs in the RPMI dataof FIG. 7B. The results of these experiments confirm that live melanomasignals were clearly and consistently differentiated. The signals wereclearly unique from those of the RPMI culture medium and the microspheretissue phantom trials. These characteristic waveforms clearly show theability of the detection system to detect malignant melanoma cells invitro.

Cultured live malignant melanoma was therefore successfully detectedusing systems and methods of the invention. The signals were clearlydifferentiated and retained a distinct pattern relating to melanomadetection. Also, when compared to the threshold trials for the examplemethods and systems discussed above in relation to testing for spheresonly, the melanoma signal was more than a 2 fold increase in voltage.

The determination of optical properties for the black latex microspheresand live melanoma cells discussed above is useful to the understandingof how the microsphere tissue phantoms compare to the live cells theyare trying to represent. Understanding how light interacts with the twoabsorbing mediums tested allows for conclusions to be drawn regardingtheir abilities to produce photoacoustic signals. Once the signalproduction potential is realized through the determination of opticalproperties, the photoacoustic response of both the melanoma cellsuspensions and the black latex microsphere suspensions can be furtherexplained. From this data correlations can be made between thesensitivity trials conducted with latex tissue phantoms and the resultsfrom actual live melanoma detection.

Integrating spheres offer a method of simultaneously determining theoptical properties of materials by employing the Inverse Adding-DoublingAlgorithm to measured light flux gathered from the integrating spheres.This discussion details the optical properties of interest, provides abrief explanation of the theory of integrating sphere measurements,describes the methodology used, and discusses the optical properties ofthe latex microspheres and malignant melanoma.

There are a number of properties that are used to describe lightinteractions with turbid media. The three properties considered indetail herein are absorption, scattering, and anisotropy. One importantoptical property for the purpose of the systems and methods of theinvention is absorption. The amount of absorption of a material at aparticular wavelength is directly related to the strength of thephotoacoustic signal that it produces.

Absorption occurs when an incident light photon interacts with amolecule that can absorb that photon in the form of molecular energytransition. In the visible spectrum these transitions consist ofelectron orbital shifts in compounds composed of conjugated dienes.Compounds containing conjugated dienes make up the majority of absorbingmaterials, or chromophores. The absorption of a compound is described byits absorption coefficient, μ_(a) defined as the probability of lightabsorption in an infinitesimal distance ds, given as cm⁻¹. Theabsorption coefficient is wavelength dependent and is determined by thetype of chromophore and its concentration. Assuming a purely absorbingmedium, the absorption coefficient can be calculated using Beer's Lawgiven as:I _(t) =I _(o) e ^(−μ) ^(a) ^(d)The absorption coefficient is exponentially related to the percentage oftransmitted light and dependent upon the thickness of the sample. Thisdescribes the simplest method of determining absorption. Realistically,a purely absorbing medium does not exist thus explaining the need formore complicated methods of optical property determination such as theinverse adding-doubling algorithm.

Scattering describes the interaction of photons that are not absorbed bya particular medium and is caused by changes in the index of refractionacross a material. Optical scattering is described by the scatteringcoefficient, t, and defined as the probability of photon scattering inan infinitesimal distance ds, given by cm⁻¹. The scattering coefficientof a material is based on the volume density of scatterers and isdependent upon the size of the particle. Beer's Law can be used in thesame manner as absorption for calculating the scattering coefficient fora purely scattering medium.

Anisotropy describes the amount of light that is forward scattered by amaterial. The phase function, or angle of light refraction, ischaracterized by anisotropy, g, which is the average cosine of the phasefunction. Anisotropy varies between isotropic scattering (g=O) andcomplete forward scattering (g=1).

There are other optical properties that may be relevant to some methodsand systems of the invention, including, for example, the scatteringalbedo given by:α=μ_(s)/(μ_(α)+μ_(s))The albedo represents the relative portion of scattering during a lightinteraction event. The total light attenuation coefficient is given by:μ_(t)=μ_(t)+μ_(s)The effective attenuation rate for highly scattered media is given by:μ_(eff)=√3μ_(α)(μ_(α)+μ_(s)(1−g))The mean free path (mfp) for a photon passing through an absorbing andscattering medium is given by:mfp=1/μ_(t)The optical depth for an absorbing and scattering medium is given by:τ=d(μ_(α)+μ_(s))Finally, the reduced scattering coefficient describes a highlyscattering medium by relating anisotropy to the scattering coefficientas:μ′_(s)=μ_(s)(1−g)All of these optical properties can be used to decipher the compositionof a material through practice of systems and methods of the invention.

These relationships can be used not only to better understand theoperation and results of systems and methods of the invention, but mayalso be used to determine and estimate qualities of detected analytes.Steps of a method of the invention include, for example, using theserelationships to determine an analyte density, mass, size,concentration, and the like.

Also, some other example methods and systems of the invention mayincludes steps and elements for building a knowledge base over time thatis useful to determine such characteristics for analytes. By way ofexample, some methods of the invention may include steps of calibrationwhereby different phantom targets (e.g., microspheres) are tested usingdifferent concentrations, different absorbance, different energy input,and other test parameter variations. A system of the invention mayinclude a memory for storing resultant data and program instructions foranalyzing the data. The resultant data can be used to develop apredictive model to use when actual test data from an unknown analyte ispresented to determine characteristics of that analyte.

Also, so called signature waveforms can be developed for differentanalytes through testing. These can be useful to identify an unknownanalyte. Further, some systems and methods of the invention may beuseful to detect and identify multiple different analytes in a singlesample. The different analytes present in the single sample can beidentified through their different waveforms.

Photoacoustic detection systems and methods of the invention have theability to detect the presence of melanin in solution. Example systemsand methods are easy to use and allow for relatively simple samplepreparation in that once a cell block is isolated, only a carrier fluidsuch as saline is required to be added in order to conduct the voltagesignal. Samples can be quickly introduced to an example system via anexternal reservoir and circulated using a pump to induce negativepressure. The test solutions can also be easily removed and the entiresystem can be cleaned in minutes providing efficient results and thecapacity for high volume processing.

Those knowledgeable in the art will appreciate that many modificationsto example systems and methods discussed above can be made. For example,detection chambers may be configured to improve upon the sensitivity ofthe device. Example systems and methods of the invention have been usedwith phantoms in the form of 6.6 μm Black CML Latex Microspheres, whichact as a broadband absorber similar to that of melanin. Photoacousticsignals derived from black latex microspheres have been discriminatedand have shown a low detection threshold as well as a very strong andclearly differentiated signal.

Methods of the invention also include steps for the isolation ofmelanoma cells from whole blood. It has been discovered that theperipheral blood mononuclear cell layer can be isolated and placed inthe detection system without creating false positives. Also, the simpleaddition of tissue phantoms in the form of latex micro spheres to wholeblood has produced strong results indicating that the protocol forsample preparation can accurately isolate foreign bodies, such asmelanoma, in the blood stream. Results have proven successful indetecting broadband absorbers in the midst of millions of mononuclearcells.

An example photoacoustic device of the invention has successfullydetected live melanoma cells in a standard saline suspension. Thephotoacoustic waveform for malignant melanoma is markedly different fromthat of other absorbers including the tissue phantom microspheres.Acoustic diffraction by the melanoma cells results in a uniquelyidentifiable photoacoustic waveform that can be used to differentiate amelanoma signal from potential false positives such as blood. Detectionthreshold is of a useful level to provide very early detection of CTC's,pathogens, and other analytes of interest. In addition, modifications tothe some elements of the systems and methods described herein arecontemplated to increase sensitivity. Such modifications include, forexample, increasing the incident beam intensity or size, decreasingirradiation area, further improving signal strength, and the like.

FIGS. 8 and 9, for example, schematically illustrate one examplealternate configuration of a flow cell 250. The cell 250 includes upperand lower sections 252 that are generally funnel shaped. A cylindricalshaped narrow throat section 254 is located between and connects the twofunnel sections 252. The throat section is made of a transparentsidewall that may be glass, by way of example. A laser 256 directs apulsed beam 258 through the throat section 256. As best shown by theoverhead view of FIG. 9, the pulsing beam 258 extends acrosssubstantially the entire diameter of the cylindrical throat section 254whereby an entire cross section of the test sample being communicatedthrough the throat section 254 is illuminated. Put another way, thisconfiguration, like that of FIGS. 3-5, captures the entire flow of thetest sample within the beam path.

An acoustic sensor 260 is arranged on a side of the throat section 254.The sensor 260 may be a film that is deflected when an acoustic wavestrikes it, or may be another device. As shown in FIGS. 8 and 9, thesensor 260 is arranged on the throat section 254 at a location adjacentto the path of the beam 258, but is located on a side where the beamdoes not directly pass to avoid (or at least minimize) potentialinterference with or from the laser beam 258.

The flow cell 250 may be dimensioned as desired and as is suitable for aparticular application. In one example configuration, the cell is in amicro scale so that the throat section 254 transports only a fewmelanoma cells and guarantees excitation of all material passing throughthe section 254. The throat section 254 may be, for example, a capillarytube having a diameter of about 1 mm or about 2 mm. If provided in asuitably small diameter, only one or a few CTC's would be expected to bein the small volume resident in the throat section 254. Directing theenergy beam 258 at the narrow throat 254 would then excite only a singeor a few CTC's at a time.

Using a configuration such as the flow cell 250 may be useful in somemethods of the invention to estimate analyte concentration. Wave spikesin the resultant data can be counted to estimate the number of analytecells detected in the sample. This knowledge together with the volume ofthe sample will lead to a determination of the analyte concentration inthe sample.

The flow cell 250 may be placed in line with a reservoir, a pump andother elements as desired. It may be used in a circulating configurationwherein a sample is passed through the throat section 254 multipletimes, or it may be used in a single pass configuration wherein a samplepasses through the flow cell 250 only a single time. In one exampleconfiguration, the flow cell 250 is used in a gravity flow arrangementthat does not rely on a pump. Or, a syringe can be used to deliver atest sample in line with the flow cell 250, with the ejecting pressurefrom the syringe urging the test sample through the cell 250. Acontroller 262 (FIG. 8) is provided and linked to both the laser 256 andto the acoustic sensor 260. The controller may be a processor baseddevice such as a computer, and includes data acquisition processing,data storage, laser control, and acoustic sensor controlfunctionalities. It can also include a display for displaying data.

As discussed above, embodiments of the invention are not limited todetection methods and systems that rely on piezoelectric photoacousticdetection. One example alternative is measurement of an opticaldisturbance that results from a photoacoustic expansion. In someapplications, it has been discovered that an increase in signal-to-noiseratio can be achieved based on detection of stress wave-induced changesusing an optical transducer. The transducer measures optical reflectanceof a glass-water interface probed with a continuous laser beam that isincident at an angle close to the critical angle of total internalreflection. A detection threshold is achieved of the order of oneindividual cell. This represents a surprising and important result.Resolution is the minimum difference between two melanoma cellconcentrations that can be distinguished by the photoacoustic sensor.

To further illustrate this embodiment of the invention, an examplesystem and method are described. Black polystyrene latex microspheres(2-BK-7000, Interfacial Dynamics Corp.,) with a diameter of 10 μm wereemployed as melanoma tissue phantoms. Microspheres were suspended in 10mL of Tyrode's buffer (125 mM NaCl, 4.7 mM KCl, 1.4 mM CaCl₂, 20 mMNaHCO₃, 0.4 mM NaH₂PO₄, 1.0 mM MgCl₂, 10 mM D-glucose, pH 7.4) resultingin concentrations ranging from 5 μspheres/μL to 200 μspheres/μL. Humanmalignant melanoma cell line Hs 936.T (C1) (ATCC) was cultured insuspension with RPMI-1640 growth medium (Sigma), incubated at 37° C.,and were suspended in 10 mL of Tyrode's buffer, concentrations were instatic conditions.

The example apparatus shown generally at 300 for this embodiment of aphotoacoustic detector of biological microparticles is schematicallyshown in FIGS. 10 and 11. An excitation laser 302, with one suitableexample being a frequency tripled Nd: YAG laser pumping an opticalparametric oscillator (Vibrant 355 II, Opotek, Carlsbad, Calif.) wasemployed to provide 500 nm laser light with a pulse duration of 10 nsthrough an optical fiber 304. Excitation beam energy entering a testchamber 306 was 6.8 mJ, with a pulse repetition rate of 10 Hz. The testchamber 306 is illustrated in FIG. 10 in isolation without upstream ordownstream connections for simplicity of illustration. Also, FIG. 10 isnot drawn to scale—relative sizes and dimensions have been altered forease of illustration.

An in vitro apparatus including a flow test chamber such as the system300 and cell 306 offer some important benefits and advantages over an invivo system in some applications. For example, an in vitro system isless intrusive on patients and their schedules. A small sample can bedrawn from a patient for testing at a later time—the patient need not bepresent and is not required to wait while the test is performed. Bodilyfluid samples can be diluted, concentrated or otherwise processed beforetesting as desired. Concentrating a sample can be useful to increase theeffective concentration of the analyte, and diluting a sample can beuseful to lower the effective analyte concentration. Each process may bedesirable in different applications, and neither is possible whentesting in vivo. Other advantages of in vitro methods and systems willbe apparent to those knowledgeable in the art.

The optical fiber 304 has a diameter of 1 mm. Other diameters will beuseful in practice of other example embodiments of the invention and maybe selected based on test chamber dimension and other designconsiderations. Also, other embodiments may orient the excitation laser302 such that no optical fiber is required. The test chamber 306 hasdimensions of about diameter 2 mm and height 1 mm, and althoughillustrated schematically in FIGS. 10 and 11 may be configured generallyas shown for cell 250 above to accommodate a flowing test sample fluid.Radiant exposure in the chamber 306 was about 0.395 J/cm², with a spotsize of about 0.74 mm radius. Other dimensions will be useful in otherapplications and embodiments. Examples are the test chambers illustratedherein above, including but not limited to cell 250 and the cell shownin FIG. 4.

In this example system 300, acoustic pressure waves were detected by asensor shown generally at 308 that comprises an optical stresstransducer. A detection electromagnetic energy beam, with examplesincluding laser or other light, is directed at an interface between twotransparent materials. The detection beam is reflected off the interfaceto a receiver. One of the transparent materials is configured to carryan acoustic pressure wave resulting from the acoustic expansion thatresults when the excitation beam impacts the absorber in the testsample. When an acoustic wave travels through the transparent liquid andreaches the interface with the transparent solid, the refractive indexchanges at the interface due to the increased density in the acousticwavefront. The change in the refractive index disrupts the reflectedbeam being received by the detector. The measurement of this disruptioncan be used to signal the presence of an acoustic expansion, andtherefore the presence of an absorber in the test sample (e.g., apathogen).

Turning now to one example of such a detector 308, as best shown in FIG.11 a thin (about 1 mm) layer of water 310 is sandwiched between the topwall 312 of a glass prism 314 and the flow chamber 306, and a flowchamber top sidewall 316 partially defines the chamber 306 and isolatesit from the fiber 304. The top sidewall 316 may be a thin glass layer orother suitable transparent material (with an example being a transparentpolymer layer) for passing light and retaining liquid. The excitationlaser beam may be introduced through sidewalls other than the top wall316.

A chamber lower sidewall made of a thin (thickness about 16 micron)plastic diaphragm 318 separates the water layer 310 from the flowchamber 306 and partially defines the chamber. The diaphragm 318 shouldbe sufficiently responsive to communicate the acoustic wave from thetest chamber 306 to the water layer 310. Fluids other than water can beused in this layer 310, although in many applications they should behighly transparent and have a viscosity similar to water. Alcohol is oneexample alternative.

A HeNe detection laser beam 320 is generated by a detector laser source321 and directed into the right angle prism 314 and reflected at theprism surface 312 underlying the water layer 310. Other energy sourcesand laser beams may also be used. A lens 324 (FIG. 10) with a focallength of 120 mm is used to focus the beam 320 to an elliptical spot onthe prism top wall 312 and water layer 310 interface. Light other than alaser beam 320 could be used, with the result that laser source 321could be a light source other than a laser. Lasers are convenient,however, for their ability to deliver a highly concentrated beam.

Referring again to FIG. 10, a second lens 326, which may be a notchlens, images the resulting HeNe laser spot on the prism surface onto theactive area of a receiver 328, with an example receiver being aphotodiode. One photodiode useful for practice of the invention is a Si1-GHz Photo-receiver, 1601-FS-AC, from NEW FOCUS, San Jose, Calif. Otherdetectors 328 may be used, other lenses than the examples 324 and 326may be used, and some embodiments may not utilize one or both lenses 324or 326. Other receivers 328 may also be used. The particular lenses (orlens combinations) and receiver may be selected on the basis ofapparatus dimensions, geometry, energy beam, and similar designparameters.

In the example embodiments of FIGS. 10-11, the laser 320 irradiates thetest chamber 306 opposite from the prism surface 312 with laser pulses.When an absorber in the test chamber absorbs energy and expands thencontracts, an acoustic wave is generated in the test sample. It iscommunicated to the thin water layer 310 and reaches the prism surface312. This causes the refractive index between the glass prism 314 andthin water layer 310 to change, and for the beam 320 being reflected tothe receiver 328 to change. The change in refractive index will causethe critical angle of reflection to change, and if the angle ofincidence of the beam 320 on the prism surface 312 is sufficiently closeto the critical angle for total reflection to briefly occur. Thismomentarily and significantly disrupts the beam 320 being reflected tothe receiver 328.

The photoacoustic signals from optical stress transducer detected by thereceiver 328 can be processed, stored on a memory, and/or displayed by acomputer 330 or other device such as an oscilloscope (with an examplebeing a 200 MHz TDS 2034B, TEKTRONIX, Wilsonville, Oreg.) triggered by aphotodiode (DET10A, Thorlabs). In the example system, the signals wereamplified with a gain of 125 via a four channel 350 MHz amplifier(SR445A, STANFORD RESEARCH SYSTEMS, Stanford, Calif.). The computer 330may include the functionality of an oscilloscope and/or othercomponents. Other amplifiers, amplification and other settings can beuseful in various embodiments of the invention. The system 300 may becontained within a cabinet 332 for convenience. The cabinet 332 mayinclude ports for bodily fluid sample entry and exit, electroniccontrols linked to various components for changing settings, and mayeven contain the computer 330 for an all-in-one system.

The prism 314 is used for convenience of configuration. It allows forthe detection laser 321 and receiver 328 to be spaced from one anotherby a predictable and useful distance. Other transparent solids can beused as alternatives to the prism 314. Examples include transparentpolymers and solids of various shapes, glass plates, glass domes, andthe like.

FIG. 12 illustrates a cross section of the effective volume of radiationin the example flow chamber 306. The effective volume of radiation isthe region where laser excitation, optoacoustic effect and detectiontake place. Since the optical fiber 304 has a numerical aperture (NA)equal to 0.37, the effective volume of radiation (V_(effe)) is a conicalsection. In these representations, θ=21.72° is the angle that correspondto NA=0.37, h is the height (i.e., distance from fiber optic cable 304and prism top wall 312) of the conical section, r is the radius of theoptical fiber 304, r+r₁ is the radius of the laser spot on the surface312, and r₁ is given by r₁=h tan θ. The effective volume of radiation isa solid of revolution obtained by the linear function ƒ(x)=tan θx+r₁,from x=0 to x=h, then

$V_{effe} = {\pi\;{h( {r^{2} + {{rh}\;\tan\;\theta} + \frac{h^{2}\tan^{2}\theta}{3}} )}}$In the example apparatus and methods, the radius and height were r=0.50mm and h=1.00 mm, respectively, resulting in V_(effe)=0.9 μL.

As discussed above, in some embodiments the laser or other beam isincident upon an entire cross section of the flowing sample so that noportion of the sample avoids irradiation as it flows through the testchamber. This can be useful to ensure that the entire test sample istested, to avoid missing any small concentration of an analyte, tospecify which particular volume of the sample contains an analyte, andfor other reasons. Further, in combination with specifying a pulse andflow rate to ensure that samples are subjected to multiple scans as theyflow through the beam, a high degree of sensitivity and accuracy can beachieved which may be particularly useful to identifying very smallconcentrations of analyte and for other purposes.

The latex microsphere solutions were introduced into the photoacousticsystem 300 to characterize the sensitivity of the device. FIG. 13illustrates the waveforms for five concentrations of microspheres andbuffer solution. The lines for the signal for the buffer solution and 2μspheres per μL are generally flat. Other lines correspond to 5, 20, 50and 100 μspheres/μL as indicated, the trials for this example embodimentresulted in a detection threshold of 5 μspheres per μL of Tyrode'sbuffer. Taking into account the total irradiated volume within thesample-glass holder it can be approximated (assuming a fully dispersedmedium) that fewer than 5 phantom melanoma cells are necessary tomaintain a strong signal during a particular data acquisition period inthis example embodiment.

As shown in FIG. 14, a linear relationship is derived between theoverall concentration of microspheres present within each test solutionand the peak excitation value. A signal-to noise ratio is used todisplay peak voltage to eliminate increased signal strength due tonoise. In this graph the filled squares are the experimental data andthe continuous line without squares is the best linear fit.

Cultured live human melanoma cells in Tyrode's buffer were introducedinto the system 300 and irradiated with 6.8 mJ of incident light energyresulting in a large photoacoustic signal around 0.8 μs. The geometry ofthe holder sample and optical detection device are conducive to thistime domain following the initial laser pulse due to a 1.5 mm/s acousticwave propagation rate in solution. Other time domains will be useful,with some dependency on the chamber 306 geometry. This time domain,however, is believed to be useful for many typical applications.

FIG. 15 illustrates resultant data. These results accurately reflect theexpected melanoma signal. A buffer solution experiment was run in orderto absolve results of false positives due to absorption by the growthmedium with resultant data shown in FIG. 15. As can been seen thiswaveform is flat, confirming that the buffer solution dose not have anyinfluence on the waveforms resulting from the melanoma cells. Again alinear relationship is obtained between the overall concentration ofmicrospheres present within each test solution and the peak excitationvalue as shown by FIG. 16. The filled squares are the experimental dataand the continuous red line is the best linear fit.

Photoacoustic detection devices and methods of the invention cansuccessfully detect small numbers of melanotic melanoma cells in a testsolution in vitro. Melanoma tissue phantoms prove the characterizationpatterns of the optical photoacoustic detector. In some embodiments ofthe invention, excitation versus concentration digresses linearly. Thisrepresents a surprising and unexpected result, and offers significantadvantages and benefits. For example, this can be useful todifferentiate between acoustic waves of different magnitudes, to allowfor the extrapolation excitation analysis, and to quantify hematogenicmelanoma concentrations for chemotherapeutic and disease progressionanalysis.

This can prove very beneficial when used in combination with an in-vitroapparatus of the invention. For example, that apparatus can be used totest a concentrated (or diluted) bodily fluid for the presence of apathogen such as melanoma. In vivo testing methods and systems do notallow for concentration or dilution of sample.

Also, in vitro testing allows for minimal invasiveness and optimalconvenience for patients as compared to in vivo. Patients need not bepresent for the testing or subject their persons to direct contact withthe apparatus. Testing can be physically and psychologically painful—invitro analysis largely eliminates these disadvantages. A blood or otherfluid sample can be drawn from the patient for later testing. The samplecan be concentrated or diluted to maximize detection capabilities.Samples can be sent to a lab for testing using an in vitro method orsystem of the invention at a high throughput efficiency. Sensitivitiesdown to a single melanoma or other pathogen cell are possible, with theresult that diseases may be identified at an early stage for treatment.

Still further, should a positive detection result the concentration ofthe pathogen in the fluid can be estimated using a system of theinvention through linear regression or other data processing step. Thisinformation can be used to estimate what stage of advancement or howextensive the presence of the pathogen is in the patient. Accordingly,some methods of the invention further include steps of building adatabase of known concentrations of melanoma and/or spheres to determinea linear data regression. Systems of the invention can include dataincluding such a database and linear regression (or other predictivemodel) stored on a memory. The computer 330 can use the regression (orother model) data to determine the concentration of anlyte (e.g.,pathogen such as melanoma cells or other) in the test sample, and outputthis information to a display for displaying or to a memory for storing.It will be appreciated that the term computer is intended to be broadlyinterpreted as a processor based device. A processor based computer orcontroller may be in any of a number of forms useful for practice withthe invention.

When using a system of the invention such as system 300 to estimatepathogen concentration or in other applications for detecting pathogens,residence time of the sample in the test chamber 306 and in particularwithin the effective volume of radiation should be considered. Asdiscussed herein above, the system 300 and test chamber 306 may beconfigured for analysis of in-vitro samples that are flowing through thetest chamber 306. By way of further illustration, FIG. 17 is a schematicshowing the example system 300 configured for operation on flowingsamples. A supply reservoir 340, pump 342, flow control device such as avalve 344 and a waste reservoir 346 are all in communication via aconduit 348 with the system 300 (FIGS. 10-11). A cabinet 332 may containall of these elements for convenience, portability, or other reasons.Test bodily sample fluid can be introduced into the sample reservoir 340via an introduction port 350 (which can be, for example a syringe portwith valve) and urged by the pump 342 through the test chamber 306 (notshown in FIG. 17) and to the waste reservoir 346. A discharge port 352through the cabinet can be used to remove sample once tested.Alternatively, a second flow control device or valve 356 can bemanipulated to allow for looped, multiple pass flow and testing.

The test chamber 306 (FIGS. 10 and 11) may be configured to ensure adesired residence time of fluid within it to ensure sufficient testingof the sample material. It can include, for example, tapered inlets andoutlets with a larger cross section and volume between so that fluidvelocity slows as it passes through the test chamber 306 and increasesresidence time as is illustrated and discussed herein with regard tocell 250 and FIG. 4. As illustrated in that FIG. and for that cell, thetest chamber 306 may have tapered inlets and outlets with a wider crosssection therebetween to ensure sufficient residence time in the pulsedlaser beam in the chamber 306.

The residence time in the chamber 306 can be considered in relation tothe frequency of pulsing of the excitation laser 302 and other factors.It is desirable to ensure that the residence time is sufficient to allowfor at least one burst of the pulsing excitation laser to illuminate alltest samples, and may be useful to configure the system 300 to allow formultiple, with an example being at least 8, 16 or 20, pulses toilluminate the test sample. Such residence times can be assured throughmanipulation of the physical dimensions of the test chamber 306, and/orthe flow control devices 344 and/or 350, and/or the pump 342, and/or theexcitation laser 302 (FIG. 10), and/or the diameter of fiber 304. By wayof example and not limitation, the excitation pulsed laser frequency canbe set at a rate that is at least greater than the residence time, atleast four times greater than the residence time, at least eight timesgreater than the residence time, and the like. This will ensure that asthe test sample flows through the cell 306, each “batch” remainsresident in the cell for a time sufficient to allow one, two, four,eight, or other desired numbers of excitation laser bursts to occur.Greater numbers of bursts may be desirable in some examples to allow foraveraging of data scans and smoothing out noisy data.

By way of a particular example, if the excitation laser is pulsing at arate of 2 bursts per millisecond, adjusting the sample flowrate throughcontrol of pump 342 (FIG. 17), valve 344, or other factors to achieve aresidence time in the chamber 306 of at least 2 milliseconds can beuseful. This will ensure that sample is subjected to at least fourpulses (i.e., 2 per millisecond×2 milliseconds) as it flows through thechamber 306.

FIG. 18 illustrates still another example system of the invention. Thissystem is generally consistent with the system illustrated in FIG. 17set up for testing of flowing samples. Similar elements have beenidentified in FIG. 18 using 400 series element numbers as were used inFIG. 17 for convenience. Further explanation and description of theseelements is not necessary and is avoided for sake of brevity. Forexample, pump 442 may be considered to be consistent with pump 342,valves 444, 452 and 456 are consistent with valves 344, 352 and 356,etc. Like the flow system of FIG. 17, the system of FIG. 18 alsoutilizes the testing system 300.

The flow testing system of FIG. 18 includes some elements not providedin the system of FIG. 17. In particular, the system of FIG. 18 isconfigured for extracting a compact sample containing the melanoma fromthe larger test sample fluid in the reservoir 440. This can be done bydiverting only the portion of the flowing sample that contains themelanoma downstream from the detector 300 (and its test chamber 306)following detection of a melanoma. The diversion is controlled to occurwhen the melanoma is passing the diversion point so that it can becollected without necessarily collecting a significant amount of othertest sample. This can be useful, for example, to further examine theparticular melanoma detected.

A flow detecting device such as a flow meter 460 is provided to measurethe flow rate of test sample flowing through the conduit 448. Athree-way bypass valve 462 is provided downstream from the detectorsystem 300 (and its chamber 306). The three-way bypass valve isconnected to a bypass conduit 464 leading to a removable collectionchamber 466. The flowmeter 460 and bypass valve 462 are connected to thecontroller computer 468.

When a melanoma is detected by the detector 300 (in the chamber 306,FIG. 1), the computer 406 can calculate the expected time for thatmelanoma to pass the bypass valve 462. This can be calculated using theflow rate (data available from either the flow meter 460 or a flowcontrol pump 442), the cross sectional area of the conduit 448, and thelength of the conduit 448 between the test chamber 306 and the bypassvalve 462. Using this data, the time it will take the melanoma to travelfrom the test chamber 306 (within 300) to the bypass valve 462 can beestimated. The controller computer 468 performs this calculation, andwill manipulate the three way bypass valve 462 at the corresponding timeto direct flow into the removable collection cell 466 (which may bemaintained initially under full or partial vacuum).

The collection cell 466 may be very small, with an example being only afew ml, one ml, one tenth ml, 50 microliters, 10 microliters, or thelike. Likewise, the collection conduit 464 may be of a very smalldiameter to minimize the required volume of sample. The computer 468manipulates the valve 462 to cause it to remain open for a sufficienttime to collect the sample, then manipulates it to cause subsequent flowto be directed to reservoir 446.

In this manner, a system of the invention may be used to collect themelanoma or other pathogen in isolation with minimal dilutant, even whenthe melanoma or pathogen is present at an extremely low concentration inthe initial sample (e.g., in a very low concentration—perhaps only asingle cell present—in the sample reservoir 440). Once the desiredportion of the flowing fluid has been diverted to the cell 464, thecontroller computer 466 manipulates the three way bypass valve 462 todirect flow to the waste reservoir 446. Those knowledgeable in the artwill appreciate that other bypass arrangements and geometries arepossible.

The detection systems and methods of the invention for detection ofanalytes such as metastatic melanoma have proven to be successful. Withthe ability to analyze blood samples within 30 minutes without the aidof a trained histologist, an example photoacoustic detection system mayprove to be a most reliable and sensitive method of detecting cancer.This unprecedented method and system could revolutionize the field ofoncology, among others, by providing a vehicle for the early detectionof metastatic disease as well as functioning as a method for determiningthe effectiveness of chemotherapeutics. In addition, if the paralleltheory of metastasis holds true, example devices and systems will be ofgreat utility as early detectors of not only metastatic disease but anymelanotic form of cancer near its inception as well as other diseases.

As an example of advancements possible through methods and systems ofthe invention, still another example system and method embodiment isillustrated. In this embodiment, a system as described generally abovewith regards to system 300 can be used as described above to detectmelanoma or other pathogen from a particular patient at very lowconcentrations, with an example being one or a few cells. Suchsensitivity was not possible in the prior art. This new level ofsensitivity can be exploited through systems and methods of theinvention by continuing to test the patient over periods of days, weeks,months and even years to determine subsequent levels of melanoma orother pathogen in the patient. Systems of the invention may include amemory or database (within computer 330, for example) that storesconcentration data over time for particular patients. Measuring theprogress of the disease in the patient at such very low concentrations(of the order of one cell) offers previously unachievable data. Not onlycan treatment be initiated at the very earliest stages of disease onset,but further the effectiveness of treatment can be measured at theseearly stages. These method and system embodiments offer previouslyunknown advantages and benefits.

Methods and systems of the invention are not limited to cancer orpathogen detection, however. Those skilled in the art will appreciatethat systems and methods will find utility in a wide variety of otherapplications. Some additional example applications include testing todetermine if a body fluid includes a particular protein or a trace of anillegal drug. Others include detecting analytes that are present insperm.

The benefits and advantages of devices and systems of the invention areevident. Discussion herein of particular example methods and systems hasbeen made for purposes of illustrating some best modes for practicingthe invention. The scope of the invention as defined by the attachedclaims, however, is not necessarily limited to the particular elementsshown and discussed. Modifications, equivalents and alternatives to thesystems and methods will be apparent to those skilled in the art.

The invention claimed is:
 1. A system for detecting a plurality ofdifferent analytes in a single bodily fluid sample comprising: a testchamber having at least one sidewall and configured to contain at leasta portion of a flowing bodily fluid sample; at least one excitationelectromagnetic energy source configured to direct a first energy beamhaving a first wavelength but not a second wavelength, and at adifferent time to direct a second energy beam having the secondwavelength but not the first wavelength into the test chamber, the firstenergy beam inducing a first thermoelastic expansion in a first analytethat absorbs the first wavelength but not the second wavelength, thesecond beam inducing a second thermoelastic expansion in a secondanalyte that absorbs the second wavelength but not the first wavelength,the first and second beams extending across the entire cross sectionalarea of the flowing sample wherein no portion of the sample escapesillumination by the beam as it flows through the test chamber; and, asensor configured to detect said first and second thermoelasticexpansions in the flowing bodily fluid sample in the test chamber, thesensor configured to measure changes in optical reflectance that resultfrom the thermoelastic expansion of the first and second analytes and toindependently determine the presence of each of the first and secondanalytes based on the independent detection of the first and secondthermoelastic expansions.
 2. A system as defined by claim 1 wherein thesensor comprises a light source for directing a light beam at areflecting interface and a receiver for receiving the reflected lightbeam and determining that a disruption to the beam has occurred.
 3. Asystem as defined by claim 2 wherein the detector further comprises oneor more lenses for focusing the light beam, the one or more lensesarranged between the light source and the receiver.
 4. A system asdefined by claim 2 and further comprising a transparent liquid layeradjacent to the test chamber and a transparent solid interfacing withthe transparent liquid layer, wherein the photoacoustic event causes theindex of refraction at the interface between the liquid layer and solidto change.
 5. A system as defined by claim 4 wherein the transparentsolid comprises a glass prism, the transparent liquid layer on a firstside of the prism, the detector light source directing the light beaminto a second side of the prism towards the prism first side, and thereflected light beam passing through a third sidewall of the prism andto the sensor.
 6. A system as defined by claim 1 wherein the sensorfurther comprises a detection laser beam source, and further comprisinga water layer adjacent to the test chamber and a glass layer interfacingwith the water layer, the glass layer reflecting the laser beam from thelaser beam source.
 7. A system as defined by claim 1 wherein the sensorfurther comprises a source for directing a light beam towards the flowchamber, a liquid layer adjacent to the flow chamber, and a detector forreceiving the detector light beam after reflection at a surface of theliquid layer.
 8. A system as defined by claim 7 wherein the sensor lightsource is arranged to cause the light beam to be incident on the liquidlayer at an angle close to the critical angle of total reflection.
 9. Asystem as defined by claim 1 and further including a computer having amemory with linear regression data stored on the memory, the computercommunicating with the sensor, the computer configured to receive adetection signal from the sensor and use the data on the memory tocalculate a concentration level of material in the bodily fluid sampleand to communicate that concentration level to one or more of a displayfor displaying and to a memory for storing.
 10. A system as defined byclaim 1 wherein: the excitation electromagnetic energy source comprisesa laser pulsing at a frequency faster than a millisecond; and, thebodily fluid sample has a residence time in the test chamber sufficientto cause the sample to be illuminated by at least 10 pulses of thelaser, all of the pulses occurring at only a single wavelength.
 11. Asystem as defined by claim 1 and further comprising: a reservoir, aconduit, and a pump, the reservoir communicating with the test chambervia the conduit, the pump arranged along the conduit to urge the samplefrom the reservoir to the test chamber and configured to cause the firstand second analytes to flow at a first flow rate and cause a firstresidence time of first and second analytes in the test chamber; and,wherein the excitation energy source comprises a pulsed laser operatingat a frequency that is sufficiently faster than the first residency timeto cause the flowing sample contained in the chamber to be illuminatedat least 20 times while it is in the test chamber, and that repeatedlyilluminates the sample with the first and second beams.
 12. A system asdefined by claim 1 wherein the excitation energy source comprises a beamhaving a diameter that extends across all of the diameter of the samplein the test chamber so that no portion of the sample escapesillumination by the beam.
 13. A system as defined by claim 1 wherein thesystem is configured to test a sample as it flows through the testchamber, the system further comprising: a downstream conduit forcarrying analyte fluid from the test chamber to a reservoir; a bypassprovided along the conduit for bypassing a portion of the sample thatcontains a pathogen to a collection cell; and, a controller for causingonly a portion of the sample that contains the pathogen to be directedthrough the bypass to the collection cell.
 14. A system as defined byclaim 13 wherein the controller comprises a computer with executableinstructions that determine the period of time it will take for thesample to reach the bypass and is configured to cause the bypass toremain open to collect a small portion of the sample that contains thepathogen.
 15. An in vitro flow system for detecting one or morecirculating cancer cells comprising: a test chamber in communicationwith a conduit; a pump urging a bodily fluid sample to flow through theconduit and through the test chamber, a pulsing excitation laser sourcethat directs a laser beam into the test chamber and illuminates anentire cross section of the flowing bodily fluid sample wherein noportion of the sample escapes illumination as it flows through the testchamber and operating at a frequency that is greater than the residencetime of the bodily fluid sample in the test chamber; a sensorcomprising: a transparent solid sandwiching a thin water layer betweenit and the test chamber; a detection laser source directing a laser beaminto the transparent solid and reflecting from the interface of thesolid and thin water layer; a receiver that receives the laser beamreflected from the transparent solid and water interface and thatmeasures a disturbance in the reflected laser beam when a change in therefractive index of reflection occurs when a wave travels through thethin water layer; and, a controller linked to the sensor for receivingwaveforms from the sensor resulting from thermoelastic expansion of theone or more cancer cells, the controller having at least a processor anda memory, the memory having a plurality of known waveforms storedthereon, the processor operative to compare a waveform received from thesensor to the stored plurality of recorded waveforms to identify anunknown cancer when its waveform matches one of the stored waveforms.16. An in-vitro flow system as defined by claim 15, wherein the lasersource directs only a single wavelength beam into the test sample, andwherein absorption of the beam causes an analyte to expand and togenerate the wave, and wherein detection of the wave determines thepresence of the analyte without requiring any additional beams withdiffering wavelengths.
 17. A system as defined by claim 1 and furthercomprising a controller having a processor, display and a memory andthat receives signals from the sensor, the memory having a plurality ofsignature waveforms stored thereon each corresponding to thermoelasticexpansions of known analytes, at least one of the signature waveformscorresponding to a first signal generated from the first thermoelasticexpansion, and at least a second of the signature waveformscorresponding to a second waveform generated from the secondthermoelastic expansion, wherein the controller identifies the first andsecond analytes in the sample based on resulting waveforms communicatedfrom the sensor.
 18. A system as defined by claim 1 wherein the sensorcan detect expansion waveforms resulting from expansions of 6.6 micronmicrospheres in a saline solution at a concentration of 7.1×10⁶microspheres/ml or less.